1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment

ABSTRACT

The invention provides antibody-drug conjugates comprising an antibody conjugated to a 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer drug moiety via a linker, and methods of using the antibody-drug conjugates.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2014/070637, filed 16 Dec. 2014, which application claims thebenefit of priority to U.S. Provisional Application Ser. No. 61/916,388filed on 16 Dec. 2013 and U.S. Provisional Application Ser. No.61/969,499 filed on 24 Mar. 2014, and to International ApplicationPCT/US2014/042560 filed on 16 Jun. 2014, all of which applications arehereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 14, 2016, isnamed P05625_US_5_Sequence_Listing.txt and is 43,470 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to antibodies conjugated to1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer drug moietiesto form antibody-drug conjugates with therapeutic or diagnosticapplications. The antibodies may be engineered with free cysteine aminoacids, reactive for conjugation with CBI dimer drug-linkerintermediates. The invention also relates to methods of using the CBIdimer antibody-drug conjugate compounds for in vitro, in situ, and invivo diagnosis or treatment of mammalian cells, or associatedpathological conditions.

BACKGROUND OF THE INVENTION

Antibody drug conjugates (ADC) are targeted chemotherapeutic moleculescombining the properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells,internalization, and release of drug, thereby enhancing their anti-tumoractivity (Carter, P. and Senter, P. (2008) The Cancer Jour.14(3):154-169). Successful ADC development for a given target antigendepends on optimization of antibody selection, linker design andstability, cytotoxic drug potency and mode of drug and linkerconjugation to the antibody (Polakis, P. (2005) Current Opinion inPharmacology 5:382-387).

The 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (amino CBI)class of DNA minor groove alkylators are potent cytotoxins (Atwell, etal (1999) J. Med. Chem., 42:3400), and have been utilized as effectorunits in a number of classes of prodrugs designed for cancer therapy.These have included antibody conjugates, (Jeffrey, et al. (2005) J. Med.Chem., 48:1344), prodrugs for gene therapy based on nitrobenzylcarbamates (Hay, et al (2003) J. Med. Chem. 46:2456) and thecorresponding nitro-CBI derivatives as hypoxia-activated prodrugs(Tercel, et al (2011) Angew. Chem., Int. Ed., 50:2606-2609). The CBI andpyrrolo[2,1-c][1,4]benzodiazepine (PBD) pharmacophores have been linkedtogether by an alkyl chain (Tercel et al (2003) J. Med. Chem46:2132-2151). PBD dimers, where two pyrrolo[2,1-c][1,4]benzodiazepineunits are tethered by an alkylene or alkylene-arylene chain are highlyefficient interstrand crosslinking agents that react with guanine in theDNA minor groove (Rahman et al (2009) Jour. Amer. Chem. Soc.131(38):13756-13766; Thurston et al (1994) Chem. Rev., 94:433-465; Boseet al (1992) J. Am. Chem. Soc. 114:4939-4941; Gregson et al (2004) Jour.Med. Chem. 47(5):1161-1174; U.S. Pat. Nos. 7,511,032; 7,528,126;7,557,099; 7,049,311; 7,067,511; 7,265,105) and have activity againstgram-positive bacteria (Doyle et al (2009) Jour. Antimicrob. Chemo.65(5):949-959; Hadjivassileva et al (2005) Jour. Antimicrob. Chemo.56(3):513-518), human B-cell chronic lymphocytic leukemia (CLL) cells(Pepper et al (2004) Cancer Res. 64(18):6750-6755), and solid tumors(Hochhauser et al (2009) Clin. Cancer Res. 15(6):2140-2147; Alley et al(2004) 64(18):6700-6706; Hartley et al (2004) Cancer Res.64(18):6693-6699). Dimeric forms of PBD have been linked to antibodiesto form ADC (US 2009/304710; US 2010/047257; US 2009/036431; WO2011/130598, WO 2011/130616; US 2013/0028919).

SUMMARY

The invention includes 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole(CBI) dimer drug moieties covalently attached by a linker to formantibody-drug conjugate (ADC) compounds with therapeutic or diagnosticapplications.

An aspect of the invention is an antibody-drug conjugate compound havingthe formula:Ab-(L-D)_(p)

wherein:

Ab is an antibody;

L is a linker having the formula:-Str-(Pep)_(m)-(Sp)_(n)-

where Str is a stretcher unit covalently attached to the antibody; Pepis an optional peptide unit of two to twelve amino acid residues, Sp isan optional spacer unit covalently attached to a dimer drug moiety, andm and n are independently selected from 0 and 1;

p is an integer from 1 to 8;

D is the dimer drug moiety having the formula:

where

R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R^(a) and R^(b) are independently selected from H and C₁-C₆ alkyloptionally substituted with one or more F,

or R^(a) and R^(b) form a five or six membered heterocyclyl group;

T is a tether group selected from C₃-C₁₂ alkylene, Y, (C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₁-C₆ alkylene)-Y—(C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₂-C₆ alkenylene)-Y—(C₂-C₆ alkenylene),and (C₂-C₆ alkynylene)-Y—(C₂-C₆ alkynylene);

where Y is independently selected from O, S, NR¹, aryl, and heteroaryl;

where alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃, N(CH₃)₂,OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally substituted withone or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with a bond to L;

D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

X¹ and X² are independently selected from O and NR³, where R³ isselected from H and C₁-C₆ alkyl optionally substituted with one or moreF;

R⁴ is H, CO₂R, or a bond to L, where R is C₁-C₆ alkyl or benzyl; and

R⁵ is H or C₁-C₆ alkyl.

An aspect of the invention is a pharmaceutical composition of theantibody-drug conjugate compound, and a pharmaceutically acceptablecarrier, glidant, diluent, or excipient.

An aspect of the invention is a method of treating cancer comprisingadministering to a patient a therapeutically-effective amount of theantibody-drug conjugate compound.

An aspect of the invention is a kit for treating cancer, comprising:

a) the pharmaceutical composition; and

b) instructions for use.

An aspect of the invention is a linker-drug intermediate selected from:X-L-D

wherein:

X is a reactive functional group selected from maleimide, thiol, amino,bromide, bromoacetamido, iodoacetamido, p-toluenesulfonate, iodide,hydroxyl, carboxyl, pyridyl disulfide, and N-hydroxysuccinimide;

L is a linker having the formula:-Str-(Pep)_(m)-(Sp)_(n)-

where Str is a stretcher unit covalently attached to X; Pep is anoptional peptide unit of two to twelve amino acid residues, Sp is anoptional spacer unit covalently attached to a dimer drug moiety, and mand n are independently selected from 0 and 1;

D is the dimer drug moiety having the formula:

where

R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R^(a) and R^(b) are independently selected from H and C₁-C₆ alkyloptionally substituted with one or more F, or R^(a) and R^(b) form afive or six membered heterocyclyl group;

T is a tether group selected from C₃-C₁₂ alkylene, Y, (C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₁-C₆ alkylene)-Y—(C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₂-C₆ alkenylene)-Y—(C₂-C₆ alkenylene),and (C₂-C₆ alkynylene)-Y—(C₂-C₆ alkynylene);

where Y is independently selected from O, S, NR¹, aryl, and heteroaryl;

where alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃, N(CH₃)₂,OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally substituted withone or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with a bond to L;

D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

X¹ and X² are independently selected from O and NR³, where R³ isselected from H and C₁-C₆ alkyl optionally substituted with one or moreF;

R⁴ is H, CO₂R, or a bond to L, where R is C₁-C₆ alkyl or benzyl; and

R⁵ is H or C₁-C₆ alkyl.

An aspect of the invention is a process for making an antibody-drugconjugate by conjugating the antibody to a linker-drug intermediate.

An aspect of the invention is a CBI dimer drug moiety compound havingthe formula:

where

R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b);

R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b);

R^(a) and R^(b) are independently selected from H and C₁-C₆ alkyloptionally substituted with one or more F,

or R^(a) and R^(b) form a five or six membered heterocyclyl group;

T is a tether group selected from C₃-C₁₂ alkylene, Y, (C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₁-C₆ alkylene)-Y—(C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₂-C₆ alkenylene)-Y—(C₂-C₆ alkenylene),and (C₂-C₆ alkynylene)-Y—(C₂-C₆ alkynylene);

where Y is independently selected from O, S, NR¹, aryl, and heteroaryl;

where alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃, N(CH₃)₂,OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally substituted withone or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with a bond to L;

D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

X¹ and X² are independently selected from O and NR³, where R³ isselected from H and C₁-C₆ alkyl optionally substituted with one or moreF;

R⁴ is H, CO₂R, where R is C₁-C₆ alkyl or benzyl; and

R⁵ is H or C₁-C₆ alkyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(2-bromo-N-methylacetamido)ethyl(methyl)carbamate 51 from(S)-tert-Butyl 1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a.

FIG. 2 shows the synthesis ofN—((R)-1-(chloromethyl)-3-(5-((R)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide53 from (R)-tert-butyl1-(chloromethyl)-5-(diphenylmethyleneamino)-1H-benzo[e]indole-3(2H)-carboxylate53a.

FIG. 3 shows the synthesis of1-((S)-5-amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53j from(S)-5-(5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57c.

FIG. 4 shows the synthesis of unnatural enantiomer,1-((R)-5-amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-((R)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53p from (R)-tert-butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 53k.

FIG. 5 shows the synthesis ofN-(4-(((S)-1-(chloromethyl)-3-(6-((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)hexanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide54 from(S)-(2-amino-4-hydroxy-5-methoxyphenyl)(2-(hydroxymethyl)pyrrolidin-1-yl)methanone54a.

FIG. 6 shows the synthesis ofN—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide55 from (S)-tert-butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a.

FIG. 7 shows the synthesis of (S)-tert-butyl8-(6-((S)-5-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate56d from (S)-tert-Butyl8-(6-((S)-1-(chloromethyl)-5-(4-nitrobenzyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-1-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate54g.

FIG. 8 shows the synthesis ofN—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(6-((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)hexanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide56 from (S)-tert-butyl8-(6-((S)-5-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate56d.

FIG. 9 shows the synthesis of(S)-5-(1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57d from (S)-tert-Butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a.

FIG. 10 shows the synthesis of(S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57i from 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid.

FIG. 11 shows the synthesis of(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57 from (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57i.

FIG. 12 shows the synthesis of(S)-3-(5-((S)-5-(4-aminobenzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58e from (S)-tert-butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a.

FIG. 13 shows the synthesis of(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58 from(S)-3-(5-((S)-5-(4-aminobenzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58e.

FIG. 14 shows the synthesis of(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 59 from (S)-tert-butyl5-(4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate55e.

FIG. 15 shows the synthesis of 2-(pyridin-2-yldisulfanyl)ethyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate61 from (S)-tert-butyl 5-amino-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate 61a.

FIG. 16 shows the synthesis of 2-(pyridin-2-yldisulfanyl)propyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate62 from 51a.

FIG. 17 shows the synthesis of(S)-3-(6-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-amino-2-methoxyphenoxy)hexanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 65d from (S)-2,2,2-trichloroethyl6-(5-(tert-butoxycarbonylamino)-4-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)hexanoate54c.

FIG. 18 shows the synthesis of benzyl alcohol lysine 65g from(S)-2-(allyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanoic acid65e.

FIG. 19 shows the synthesis of(S)-4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanamido)-6-(tert-butoxycarbonylamino)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate65k from(S)-3-(6-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-amino-2-methoxyphenoxy)hexanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 65d.

FIG. 20 shows the synthesis of(S)-4-((S)-6-amino-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate65 as the bis-trifluoroacetate salt from 65k.

FIG. 21 shows the synthesis of (S)-di-tert-butyl1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl phosphate 66d from(S)-tert-butyl5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate 57a,prepared from 51a.

FIG. 22 shows the synthesis ofN-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-phosphonoxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide66 from (2E,2′E)-tert-butyl 3,3′-(2-nitro-1,4-phenylene)diacrylate 66e.

FIG. 23 shows the synthesis ofN-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide67 from (2E,2′E)-tert-butyl3,3′-(2-(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)propanamido)-1,4-phenylene)diacrylate66g.

FIG. 24 shows the synthesis of(S)-1-(chloromethyl)-3-((E)-3-(4-((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)-2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)phenyl)acryloyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 68 from 66d, 67c, 67d.

FIG. 25 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 3 days versus concentrations (μg/ml)of Thio hu anti-CD22 HC A121C-MC-vc-PAB-(CBI dimer) 101 and Thio huanti-Her2 HC A121C-MC-vc-PAB-(CBI dimer) 102.

FIG. 26 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 3 days versus concentrations (μg/ml)of Thio Hu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI-PBD) 103 and Thio HuAnti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI-PBD) 104

FIG. 27 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 3 days versus concentrations (μg/ml)of Thio Hu Anti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI dimer) 116 and ThioHu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI dimer) 117.

FIG. 28 shows the efficacy of antibody-drug conjugates in a plot ofEOL-1 in vitro cell viability at 3 days versus concentrations (μg/ml) ofThio Hu Anti-CD33 15G15.33 HC A118C-MC-MMED-(CBI dimer phos) 125.

FIG. 29 shows the efficacy of antibody-drug conjugates in a plot ofEOL-1 in vitro cell viability at 3 days versus concentrations (μg/ml) ofThio Hu Anti-MUC16 3A5 HC A118C-MC-ED-(CBI dimer DVB diphos) 126 andThio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBI dimer DVB diphos) 127.

FIG. 30 shows the efficacy of antibody-drug conjugates in a plot ofEOL-1 in vitro cell viability at 3 days versus concentrations (μg/ml) ofThio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBI dimer DVB diphos) 127,Thio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBI dimer DVB phos) 129, andThio Hu Anti-MUC16 3A5 HC A118C-MC-ED-(CBI dimer DVB phos) 130.

FIG. 31 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: (1) Vehicle: Histidine Buffer #8: 20 mMHistidine Acetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (2) Thio HuAnti-CD22 10F4v3 HC A118C-MC-MMED-(CBI dimer phos) 110, (3) Thio HuAnti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI dimer MePip) 108, (4) Thio HuAnti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI dimer phos) 111, (5) Thio HuAnti-Her2 4D5 HC A118C-MC-MMED-(CBI dimer phos) 109, (6) Thio HuAnti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI dimer MePip) 107, (7) Thio HuAnti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI dimer phos) 112. ADC were dosed at10 mg/kg.

FIG. 32 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: (1) Vehicle: Histidine Buffer #8: 20 mMHistidine Acetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (2) Thio HuAnti-CD22 10F4v3 HC A118C-DSE-(CBI dimer phos) 120, 10 mg/kg, (3) ThioHu Anti-CD22 10F4v3 HC A118C-DSE-(CBI dimer phos) 122, 10 mg/kg, (4)Thio Hu Anti-CD22 10F4v3 HC A118C-MC-vc-PAB-(N10,PBD-CBI MePip) 124, 10mg/kg, (5) Thio Hu Anti-Her2 4D5 HC A118C-DSE-(CBI dimer phos) 119, 3mg/kg, (6) Thio Hu Anti-Her2 4D5 HC A118C-DSE-(CBI dimer phos) 119, 10mg/kg, (7) Thio Hu Anti-Her2 4D5 HC A118C-DSP-(CBI dimer phos) 121, 3mg/kg, (8) Thio Hu Anti-Her2 4D5 HC A118C-DSP-(CBI dimer phos) 121, 10mg/kg (9) Thio Hu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(N10,PBD-CBI MePip)123, 3 mg/kg, (10) Thio Hu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(N10,PBD-CBIMePip) 123, 10 mg/kg.

FIG. 33 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in OVCAR3X2.1 human ovariantumors inoculated into C.B-17 SCID mice after dosing once IV with: (1)Vehicle: Histidine Buffer #8: 20 mM Histidine Acetate, pH 5.5, 240 mMSucrose, 0.02% PS 20, (2) Thio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBIdimer DVB diphos) 127, 3 mg/kg, (3) Thio Hu Anti-MUC16 3A5 HCA118C-MC-ED-(CBI dimer DVB diphos) 126, 3 mg/kg, (4) Thio Hu Anti-MUC163A5 HC A118C-MC-ED-(CBI dimer DVB diphos) 126, 1 mg/kg.

FIG. 34 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in HL-60 human acutemyeloid leukemia inoculated into C.B-17 SCID mice after dosing once IVat 20 μg/m2 with: (1) Vehicle: Histidine Buffer #8: 20 mM HistidineAcetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (2) Thio Hu Anti-CD3315G15.33 HC A118C-MC-MMED-(CBI dimer phos) 125, (3) Thio Hu Anti-CD3315G15.33 HC A118C-MC-ED-(CBI dimer DVB diphos) 127, (4) Thio HuAnti-MUC16 3A5 HC A118C-MC-MMED-(CBI dimer phos) 128, (5) Thio HuAnti-MUC16 3A5 HC A118C-MC-ED-(CBI dimer DVB diphos) 126.

FIG. 35a shows efficacy of ADC138 in SCID mice with HL-60 human acutemyeloid leukemia tumors. ADC138 demonstrated dose-dependent inhibitionof tumor growth compared with vehicle group. The non-targeting controlADC135 had no effect on tumor growth.

FIG. 35b shows efficacy of ADC139 in SCID mice with HL-60 human acutemyeloid leukemia tumors. ADC139 demonstrated clear inhibition of tumorgrowth compared with vehicle group. The non-targeting control ADC136 at1 mg/kg had a modest effect on tumor growth; however, ADC139 at thematching dose was substantially more effective, resulting in completetumor remission.

FIG. 36 shows efficacy of ADC134 in SCID-beige mice with Igrov-1 humanovarian tumors. ADC134 demonstrated dose-dependent inhibition of tumorgrowth compared with vehicle group. The non-targeting control ADC137 hadno effect on tumor growth.

FIG. 37 shows the synthesis ofN-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide(Compound No. 69, Table 4).

FIG. 38 shows the synthesis of 2-(pyridin-2-yldisulfanyl)propyl2,5-bis((E)-3-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylcarbamate(Table 4, Compound No. 72, FIG. 38).

FIG. 39 shows the synthesis of[(1S)-1-(chloromethyl)-3-[(E)-3-[4-[(E)-3-[(1S)-1-(chloromethyl)-5-phosphonooxy-1,2-dihydrobenzo[e]indol-3-yl]-3-oxo-prop-1-enyl]-2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]phenyl]prop-2-enoyl]-1,2-dihydrobenzo[e]indol-5-yl]dihydrogenphosphate (Compound No. 78, Table 4, FIG. 39).

FIG. 40 shows the synthesis of[(1S)-1-(chloromethyl)-3-[(E)-3-[4-[(E)-3-[(1S)-1-(chloromethyl)-5-phosphonooxy-1,2-dihydrobenzo[e]indol-3-yl]-3-oxo-prop-1-enyl]-2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]phenyl]prop-2-enoyl]-1,2-dihydrobenzo[e]indol-5-yl]dihydrogenphosphate (Compound No. 79, Table 4, FIG. 40).

FIG. 41 shows the synthesis of 2-(2-pyridyldisulfanyl)propylN-[1-(chloromethyl)-3-[5-[1-(chloromethyl)-5-hydroxy-1,2-dihydrobenzo[e]indol-3-yl]-5-oxo-pentanoyl]-1,2-dihydrobenzo[e]indol-5-yl]carbamate(Compound No. 80, Table 4, FIG. 41).

FIG. 42-43 show the synthesis of 2-(2-pyridyldisulfanyl)propyl3-[6-[1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyl)oxy-1,2-dihydrobenzo[e]indol-3-yl]-6-oxo-hexoxy]-6-hydroxy-2-methoxy-11-oxo-6a,7,8,9-tetrahydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate,2-(2-pyridyldisulfanyl)propyl3-[6-[1-(chloromethyl)-5-phosphonooxy-1,2-dihydrobenzo[e]indol-3-yl]-6-oxo-hexoxy]-6-hydroxy-2-methoxy-11-oxo-6a,7,8,9-tetrahydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate,and 2-(2-pyridyldisulfanyl)propyl3-[6-[1-(chloromethyl)-5-hydroxy-1,2-dihydrobenzo[e]indol-3-yl]-6-oxo-hexoxy]-6-hydroxy-2-methoxy-11-oxo-6a,7,8,9-tetrahydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate(Compound No. 81-83, Table 4, FIG. 42-43).

FIG. 44 shows the synthesis of(1S)-1-(chloromethyl)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(chloromethyl)-5-[(6-methyl-β-D-glucopyranuronosyl)oxy]-1,2-dihydro-3H-benzo[e]indol-3-yl}-3-oxo-1-propenyl)-2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]phenyl}-2-propenoyl)-1,2-dihydro-3H-benzo[e]indol-5-ylmethyl 3-D-glucopyranosiduronate (Compound No. 84, Table 4, FIG. 44).

FIG. 45 shows the synthesis of(S)-(1-methyl-1H-pyrrole-2,5-diyl)bis(((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)methanone)(Compound No. 15, table 1, FIG. 45).

FIG. 46 shows the synthesis ofN-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenyl)acetamide(Compound No. 16, Table 1, FIG. 46).

FIG. 47 shows the synthesis of(S,2E,2′E)-3,3′-(2-methoxy-1,4-phenylene)bis(1-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)prop-2-en-1-one)(Compound No. 17, Table 1, FIG. 47).

FIG. 48 shows the synthesis of(S,2E,2′E)-3,3′-(1-methyl-1H-pyrrole-2,5-diyl)bis(1-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)prop-2-en-1-one)(Compound No. 18 Table 1, FIG. 48).

FIG. 49 shows the synthesis of(S)-3,3′-(2-methoxy-1,4-phenylene)bis(1-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)prop-2-yn-1-one)(Compound 19, table 1, FIG. 49).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the illustrated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, and are consistent with:Singleton et al (1994) Dictionary of Microbiology and Molecular Biology,2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C., Travers, P.,Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., GarlandPublishing, New York.

Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

When trade names are used herein, applicants intend to independentlyinclude the trade name product formulation, the generic drug, and theactive pharmaceutical ingredient(s) of the trade name product.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; minibodies (Olafsen et al (2004) ProteinEng. Design & Sel. 17(4):315-323), fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, CDR(complementary determining region), and epitope-binding fragments of anyof the above which immunospecifically bind to cancer cell antigens,viral antigens or microbial antigens, single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature, 256:495, or may be made byrecombinant DNA methods (see for example: U.S. Pat. No. 4,816,567;5,807,715). The monoclonal antibodies may also be isolated from phageantibody libraries using the techniques described in Clackson et al(1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol.,222:581-597; for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape, etc.) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc constant region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact immunoglobulin antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. Ig forms includehinge-modifications or hingeless forms (Roux et al (1998) J. Immunol.161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005/0048572; US 2004/0229310).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

A “free cysteine amino acid” refers to a cysteine amino acid residuewhich has been engineered into a parent antibody, has a thiol functionalgroup (—SH), and is not paired as an intramolecular or intermoleculardisulfide bridge.

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, a linker is a divalent radical, specified as L.

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents. The term “substituent” denotes an atom ora group of atoms replacing a hydrogen atom on the parent molecule. Theterm “substituted” denotes that a specified group bears one or moresubstituents. Where any group may carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of any length from one totwelve carbon atoms (C₁-C₁₂), wherein the alkyl radical may beoptionally substituted independently with one or more substituentsdescribed below. In another embodiment, an alkyl radical is one to eightcarbon atoms (C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples ofalkyl groups include, but are not limited to, methyl (Me, —CH₃), ethyl(Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of any length from one totwelve carbon atoms (C₁-C₁₂), wherein the alkylene radical may beoptionally substituted independently with one or more substituentsdescribed below. In another embodiment, an alkylene radical is one toeight carbon atoms (C₁-C₈), or one to six carbon atoms (C₁-C₆). Examplesof alkylene groups include, but are not limited to, methylene (—CH₂—),ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of any length from two to eight carbon atoms (C₂-C₈)with at least one site of unsaturation, i.e., a carbon-carbon, sp²double bond, wherein the alkenyl radical may be optionally substitutedindependently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenyl or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and thelike.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of any length from two to eight carbon atoms (C₂-C₈)with at least one site of unsaturation, i.e., a carbon-carbon, sp²double bond, wherein the alkenylene radical may be optionallysubstituted independently with one or more substituents describedherein, and includes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenylene or vinylene (—CH═CH—), allyl (—CH₂CH═CH—), andthe like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of any length from two to eight carbon atoms (C₂-C₈) with atleast one site of unsaturation, i.e., a carbon-carbon, sp triple bond,wherein the alkynyl radical may be optionally substituted independentlywith one or more substituents described herein. Examples include, butare not limited to, ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), andthe like.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of any length from two to eight carbon atoms (C₂-C₈)with at least one site of unsaturation, i.e., a carbon-carbon, sp triplebond, wherein the alkynylene radical may be optionally substitutedindependently with one or more substituents described herein. Examplesinclude, but are not limited to, ethynylene (—C≡C—), propynylene(propargylene, —CH₂C≡C—), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Spiro moieties are also included within the scopeof this definition. Examples of monocyclic carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like. Carbocyclyl groups areoptionally substituted independently with one or more substituentsdescribed herein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted with one or more substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocyclesare described in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl,1-methyl-1H-benzo[d]imidazole, [1,2,4]triazolo[1,5-a]pyridine,pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl,triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionallysubstituted independently with one or more substituents describedherein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or 3-carboline.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and l or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of anantibody-drug conjugate (ADC). Exemplary salts include, but are notlimited, to sulfate, citrate, acetate, oxalate, chloride, bromide,iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate,lactate, salicylate, acid citrate, tartrate, oleate, tannate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucuronate, saccharate, formate, benzoate,glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counterion. The counterion may beany organic or inorganic moiety that stabilizes the charge on the parentcompound. Furthermore, a pharmaceutically acceptable salt may have morethan one charged atom in its structure. Instances where multiple chargedatoms are part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counterion.

The following abbreviations are used herein and have the indicateddefinitions: BME is beta-mercaptoethanol, Boc is N-(t-butoxycarbonyl),cit is citrulline (2-amino-5-ureido pentanoic acid), DCC is1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, DMA is dimethylacetamide, DMAP is4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO isdimethylsulfoxide, DTT is dithiothreitol, EDCI is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl(or 4-methoxytrityl), NHS is N-hydroxysuccinimide, PBS isphosphate-buffered saline (pH 7), PEG is polyethylene glycol or a unitof ethylene glycol (—OCH₂CH₂—), Ph is phenyl, Pnp is p-nitrophenyl, MCis 6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromotris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusionchromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC isthin layer chromatography, UV is ultraviolet, and val is valine.

Cysteine Engineered Antibodies

The compounds of the invention include antibody-drug conjugatescomprising cysteine engineered antibodies where one or more amino acidsof a wild-type or parent antibody are replaced with a cysteine aminoacid. Any form of antibody may be so engineered, i.e. mutated. Forexample, a parent Fab antibody fragment may be engineered to form acysteine engineered Fab, referred to herein as “ThioFab.” Similarly, aparent monoclonal antibody may be engineered to form a “ThioMab.” Itshould be noted that a single site mutation yields a single engineeredcysteine residue in a ThioFab, while a single site mutation yields twoengineered cysteine residues in a ThioMab, due to the dimeric nature ofthe IgG antibody. Mutants with replaced (“engineered”) cysteine (Cys)residues are evaluated for the reactivity of the newly introduced,engineered cysteine thiol groups. The thiol reactivity value is arelative, numerical term in the range of 0 to 1.0 and can be measuredfor any cysteine engineered antibody. Thiol reactivity values ofcysteine engineered antibodies of the invention are in the ranges of 0.6to 1.0; 0.7 to 1.0; or 0.8 to 1.0.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Doman et al(2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; 7,723,485;WO2009/052249, Shen et al (2012) Nature Biotech., 30(2):184-191;Junutula et al (2008) Jour of Immun. Methods 332:41-52). The engineeredcysteine thiols may react with linker reagents or the linker-drugintermediates of the present invention which have thiol-reactive,electrophilic groups such as maleimide or alpha-halo amides to form ADCwith cysteine engineered antibodies (ThioMabs) and the drug (D) moiety.The location of the drug moiety can thus be designed, controlled, andknown. The drug loading can be controlled since the engineered cysteinethiol groups typically react with thiol-reactive linker reagents orlinker-drug intermediates in high yield. Engineering an antibody tointroduce a cysteine amino acid by substitution at a single site on theheavy or light chain gives two new cysteines on the symmetricalantibody. A drug loading near 2 can be achieved and near homogeneity ofthe conjugation product ADC.

Cysteine engineered antibodies of the invention preferably retain theantigen binding capability of their wild type, parent antibodycounterparts. Thus, cysteine engineered antibodies are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor-associated antigens (TAA), cell surface receptorproteins and other cell surface molecules, transmembrane proteins,signaling proteins, cell survival regulatory factors, cell proliferationregulatory factors, molecules associated with (for e.g., known orsuspected to contribute functionally to) tissue development ordifferentiation, lymphokines, cytokines, molecules involved in cellcycle regulation, molecules involved in vasculogenesis and moleculesassociated with (for e.g., known or suspected to contribute functionallyto) angiogenesis. The tumor-associated antigen may be a clusterdifferentiation factor (i.e., a CD protein). An antigen to which acysteine engineered antibody is capable of binding may be a member of asubset of one of the above-mentioned categories, wherein the othersubset(s) of said category comprise other molecules/antigens that have adistinct characteristic (with respect to the antigen of interest).

Cysteine engineered antibodies are prepared for conjugation withlinker-drug intermediates by reduction and reoxidation of intrachaindisulfide groups (Example 19).

Cysteine engineered antibodies which may be useful in the antibody-drugconjugates of the invention in the treatment of cancer include, but arenot limited to, antibodies against cell surface receptors andtumor-associated antigens (TAA). Tumor-associated antigens are known inthe art, and can be prepared for use in generating antibodies usingmethods and information which are well known in the art. In attempts todiscover effective cellular targets for cancer diagnosis and therapy,researchers have sought to identify transmembrane or otherwisetumor-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Examples of tumor-associated antigens TAA include, but are not limitedto, TAA (1)-(51) listed below. For convenience, information relating tothese antigens, all of which are known in the art, is listed below andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA (1)-(51) areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

Tumor-Associated Antigens:

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_001203)

ten Dijke, P., et al Science 264 (5155):101-104 (1994), Oncogene 14(11):1377-1382 (1997)); WO2004063362 (Claim 2); WO2003042661 (Claim 12);US2003134790-A1 (Page 38-39); WO2002102235 (Claim 13; Page 296);WO2003055443 (Page 91-92); WO200299122 (Example 2; Page 528-530);WO2003029421 (Claim 6); WO2003024392 (Claim 2; FIG. 112); WO200298358(Claim 1; Page 183); WO200254940 (Page 100-101); WO200259377 (Page349-350); WO200230268 (Claim 27; Page 376); WO200148204 (Example; FIG.4) NP_001194 bone morphogenetic protein receptor, typeIB/pid=NP_001194.1—Cross-references: MIM:603248; NP_001194.1; AY065994(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486)Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395(6699):288-291 (1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267(16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV);WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33;Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (FIG. 3);WO2003025138 (Claim 12; Page 150);NP_003477 solute carrier family 7 (cationic amino acid transporter, y+system), member 5/pid=NP_003477.3—Homo sapiensCross-references: MIM:600182; NP_003477.3; NM_015923; NM_003486_1(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_012449)Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999) Proc.Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004065577 (Claim 6);WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (Claim 2);WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page 618-619);WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173, Example 2; FIG.2A);NP_036581 six transmembrane epithelial antigen of the prostateCross-references: MIM:604415; NP_036581.1; NM_012449_1(4) 0772P (CA125, MUC16, Genbank accession no. AF361486)J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004045553 (Claim 14);WO200292836 (Claim 6; FIG. 12); WO200283866 (Claim 15; Page 116-121);US2003124140 (Example 16); Cross-references: GI:34501467; AAK74120.3;AF361486_1(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_005823) Yamaguchi, N., et al Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol.Chem. 270 (37):21984-21990 (1995)); WO2003101283 (Claim 14);(WO2002102235 (Claim 13; Page 287-288); WO2002101075 (Claim 4; Page308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);Cross-references: MIM:601051; NP_005814.2; NM_005823_1(6) Napi2b (NAPI-2B, NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34(sodium phosphate), member 2, type II sodium-dependent phosphatetransporter 3b, Genbank accession no. NM_006424)J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284(1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004022778 (Claim 2); EP1394274 (Example 11);WO2002102235 (Claim 13; Page 326); EP875569 (Claim 1; Page 17-19);WO200157188 (Claim 20; Page 329); WO2004032842 (Example IV); WO200175177(Claim 24; Page 139-140);Cross-references: MIM:604217; NP_006415.1; NM_006424_1(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878)Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1);WO2003003984 (Claim 1); WO200206339 (Claim 1; Page 50); WO200188133(Claim 1; Page 41-43, 48-58); WO2003054152 (Claim 20); WO2003101400(Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew;HGNC:10737;(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al (2002)Cancer Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180 (Claim12); US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056(Example 5); WO2003105758 (Claim 12); US2003206918 (Example 5);EP1347046 (Claim 1); WO2003025148 (Claim 20);Cross-references: GI:37182378; AAQ88991.1; AY358628_1(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et alEur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79,1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et alHum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet.12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002)Hum. Genet. 111, 198-206; WO2004045516 (Claim 1); WO2004048938 (Example2); WO2004040000 (Claim 151); WO2003087768 (Claim 1); WO2003016475(Claim 1); WO2003016475 (Claim 1); WO200261087 (FIG. 1); WO2003016494(FIG. 6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Page124-125); EP522868 (Claim 8; FIG. 2); WO200177172 (Claim 1; Page297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No.5,773,223 (Claim 1a; Col 31-34); WO2004001004;(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accessionno. NM_017763);WO2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim12); WO2003083074 (Claim 14; Page 61); WO2003018621 (Claim 1);WO2003024392 (Claim 2; FIG. 93); WO200166689 (Example 6);Cross-references: LocusID:54894; NP_060233.2; NM_017763_1(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138)Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397(Claim 1; FIG. 1); WO200272596 (Claim 13; Page 54-55); WO200172962(Claim 1; FIG. 4B); WO2003104270 (Claim 11); WO2003104270 (Claim 16);US2004005598 (Claim 22); WO2003042661 (Claim 12); US2003060612 (Claim12; FIG. 10); WO200226822 (Claim 23; FIG. 2); WO200216429 (Claim 12;FIG. 10);Cross-references: GI:22655488; AAN04080.1; AF455138_1(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_017636)Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697(2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820(2003)); US2003143557 (Claim 4); WO200040614 (Claim 14; Page 100-103);WO200210382 (Claim 1; FIG. 9A); WO2003042661 (Claim 12); WO200230268(Claim 27; Page 391); US2003219806 (Claim 4); WO200162794 (Claim 14;FIG. 1A-D);Cross-references: MIM:606936; NP_060106.2; NM_017636_1(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_003203 or NM_003212)Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (Claim 1); WO2003083041(Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53);WO2003024392 (Claim 2; FIG. 58); WO200216413 (Claim 1; Page 94-95, 105);WO200222808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2);Cross-references: MIM:187395; NP_003203.1; NM_003212_1(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004)Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., etal J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad.Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83,5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320;WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim9); WO2004045520 (Example 4); WO9102536 (FIGS. 9.1-9.9); WO2004020595(Claim 1);Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.(15) CD79b (CD79B, CD793, IGb (immunoglobulin-associated beta), B29,Genbank accession no. NM_000626 or 11038674)Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625);WO2004016225 (claim 2, FIG. 140); WO2003087768, US2004101874 (claim 1,page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573(claim 5, page 15); U.S. Pat. No. 5,644,033; WO2003048202 (claim 1,pages 306 and 309); WO 99/558658, U.S. Pat. No. 6,534,482 (claim 13,FIG. 17A/B); WO200055351 (claim 11, pages 1145-1146);Cross-references: MIM:147245; NP_000617.1; NM_000626_1(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAPIB, SPAPIC, Genbank accession no. NM_030764,AY358130)Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95(2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. U.S.A. 98(17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem. Biophys. Res.Commun. 280 (3):768-775; WO2004016225 (Claim 2); WO2003077836;WO200138490 (Claim 5; FIG. 18D-1-18D-2); WO2003097803 (Claim 12);WO2003089624 (Claim 25);Cross-references: MIM:606509; NP_110391.2; NM_030764_1(17) HER2 (ErbB2, Genbank accession no. M11730) Coussens L., et alScience (1985) 230(4730):1132-1139); Yamamoto T., et al Nature 319,230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82,6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165, 869-880, 2004;Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., etal Nature 421, 756-760, 2003; Ehsani A., et al (1993) Genomics 15,426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I); WO2004009622;WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim 1);US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; FIG.1A-B); WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (Claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (Claim 52; FIG. 7); WO200020579(Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38);WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361(Claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); Accession:P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.(18) NCA (CEACAM6, Genbank accession no. M18728);Bamett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem.Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc.Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393(Claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (Claim12); WO200278524 (Example 2); WO200286443 (Claim 27; Page 427);WO200260317 (Claim 2);Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728;(19) MDP (DPEP1, Genbank accession no. BC017023)Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003016475(Claim 1); WO200264798 (Claim 33; Page 85-87); JP05003790 (FIG. 6-8);WO9946284 (FIG. 9);Cross-references: MIM:179780; AAH17023.1; BC017023_1(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et alNature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001;Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J.,et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003)Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (Claim 1; Page 55-59); Accession:Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053) Gary S. C.,et al Gene 256, 139-147, 2000; Clark H. F., et al Genome Res. 13,2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A.99, 16899-16903, 2002; US2003186372 (Claim 11); US2003186373 (Claim 11);US2003119131 (Claim 1; FIG. 52); US2003119122 (Claim 1; FIG. 52);US2003119126 (Claim 1); US2003119121 (Claim 1; FIG. 52); US2003119129(Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52);US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1);(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.NM_004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991)Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998),Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12);WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583(Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42);Cross-references: MIM:600997; NP_004433.2; NM_004442_1(23) ASLG659 (B7h, Genbank accession no. AX092328) US20040101899 (Claim2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3); US2003165504 (Claim1); US2003124140 (Example 2); US2003065143 (FIG. 60); WO2002102235(Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6;FIG. 10); WO200194641 (Claim 12; FIG. 7b); WO200202624 (Claim 13; FIG.1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2;Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469);WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3; Pages 190-192);WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12);WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318;(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436) Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem.Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274(Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1);WO200281646 (Claim 1; Page 164); WO2003003906 (Claim 10; Page 288);WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b);WO200032752 (Claim 18; FIG. 1); WO9851805 (Claim 17; Page 97); WO9851824(Claim 10; Page 94); WO9840403 (Claim 2; FIG. 1B);Accession: 043653; EMBL; AF043498; AAC39607.1.(25) GEDA (Genbank accession No. AY260763);AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1—Homosapiens Species: Homo sapiens (human)WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example3, Claim 20); US2003194704 (Claim 45);Cross-references: GI:30102449; AAP14954.1; AY260763_1(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. AF116456); BAFF receptor/pid=NP_443177.1—Homosapiens Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (Claim 35; FIG. 6B); WO2003035846 (Claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); WO200224909(Example 3; FIG. 3);Cross-references: MIM:606269; NP_443177.1; NM_052945_1; AF132600(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814, Genbank accession No. AK026467);Wilson et al (1991) J. Exp. Med. 173:137-146; WO2003072036 (Claim 1;FIG. 1); Cross-references: MIM:107266; NP_001762.1; NM_001771_1(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] GeneChromosome: 19q13.2, Genbank accession No. NP_001774.10) WO2003088808,US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No.5,644,033; Ha et al (1992) J. Immunol. 148(5):1526-1531; Mueller et al(1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al (1994)Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp.Immunol. 90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637;Sakaguchi et al (1988) EMBO J. 7(11):3457-3464;(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa,pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accessionNo. NP_001707.1)WO2004040000; WO2004015426; US2003105292 (Example 2); U.S. Pat. No.6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188 (Claim 20, page269); WO200172830 (pages 12-13); WO200022129 (Example 1, pages 152-153,Example 2, pages 254-256); WO9928468 (claim 1, page 38); U.S. Pat. No.5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58); WO9217497(claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol. 22:2795-2799;Barella et al (1995) Biochem. J. 309:773-779;(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+ T lymphocytes); 273 aa, pI:6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_002111.1)Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989)Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) TissueAntigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119;(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No.NP_002552.2) Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004047749;WO2003072035 (claim 10); Touchman et al (2000) Genome Res. 10:165-173;WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1);WO2003029277 (page 82);(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCEFull maeaity . . . tafrfpd (1 . . . 359; 359 aa), pI: 8.66, MW: 40225TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No. NP_001773.1)WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655(pages 105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877;Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903;(33) LY64 (Lymphocyte antigen 64 (RP 105), type I membrane protein ofthe leucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa, pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No.NP_005573.1)US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al (1996)Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822;WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages24-26);(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa, pI: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.NP_443170.1)WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2); Davis et al(2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (claim8); EP1347046 (claim 1); WO2003089624 (claim 7);(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies); 977 aa, pI: 6.88 MW: 106468 TM: 1 [P] GeneChromosome: 1q21, Genbank accession No. Human:AF343662, AF343663,AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187,AY358085; Mouse:AK089756, AY158090, AY506558; NP_112571.1WO2003024392 (claim 2, FIG. 97); Nakayama et al (2000) Biochem. Biophys.Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, FIG.18B-1-18B-2);(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBIRefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;Genbank accession No. AF179274; AY358907, CAF85723, CQ782436WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8);WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944(pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706);US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350;US2004005563; US2003124579; Horie et al (2000) Genomics 67:146-152;Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang etal (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int JCancer. October 15; 94(2):178-84;(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; (SI); (SIL); ME20;gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R. P.et al (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33), 13731-13736;Kummer, M. P. et al (2009) J. Biol. Chem. 284 (4), 2296-2306;(38) TMEFF1 (transmembrane protein with EGF-like and twofollistatin-like domains 1i Tomoregulin-1; H7365; C9orf2; C9ORF2;U19878; X83961) NM_080655; NM_003692; Harms, P. W. (2003) Genes Dev. 17(21), 2624-2629; Gery, S. et al (2003) Oncogene 22 (18):2723-2727;(39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA;RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1; U95847; BC014962;NM_145793) NM_005264; Kim, M. H. et al (2009) Mol. Cell. Biol. 29 (8),2264-2277; Treanor, J. J. et al (1996) Nature 382 (6586):80-83;(40) Ly6E (lymphocyte antigen 6 complex, locus E;Ly67,RIG-E,SCA-2,TSA-1) NP_002337.1; NM_002346.2; de Nooij-van Dalen, A.G. et al (2003) Int. J. Cancer 103 (6), 768-774; Zammit, D. J. et al(2002) Mol. Cell. Biol. 22 (3):946-952;(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2) NP_001007539.1;NM_001007538.1; Furushima, K. et al (2007) Dev. Biol. 306 (2), 480-492;Clark, H. F. et al (2003) Genome Res. 13 (10):2265-2270;(42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1)NP_067079.2; NM_021246.2; Mallya, M. et al (2002) Genomics 80(1):113-123; Ribas, G. et al (1999) J. Immunol. 163 (1):278-287;(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49, GPR67) NP_003658.1; NM_003667.2; Salanti, G. et al (2009) Am. J.Epidemiol. 170 (5):537-545; Yamamoto, Y. et al (2003) Hepatology 37(3):528-533;(44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; (PTC); CDHF12;Hs.168114; RET51; RET-ELE1) NP_066124.1; NM_020975.4; Tsukamoto, H. etal (2009) Cancer Sci. 100 (10):1895-1901; Narita, N. et al (2009)Oncogene 28 (34):3058-3068;(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348;FLJ35226) NP_059997.3; NM_017527.3; Ishikawa, N. et al (2007) CancerRes. 67 (24):11601-11611; de Nooij-van Dalen, A. G. et al (2003) Int. J.Cancer 103 (6):768-774;(46) GPR19 (G protein-coupled receptor 19; Mm.4787) NP_006134.1;NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105 (1-2):162-164; O'Dowd, B. F. et al (1996) FEBS Lett. 394 (3):325-329;(47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12)NP_115940.2; NM_032551.4; Navenot, J. M. et al (2009) Mol. Pharmacol. 75(6):1300-1306; Hata, K. et al (2009) Anticancer Res. 29 (2):617-623;(48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982)NP_859069.2; NM_181718.3; Gerhard, D. S. et al (2004) Genome Res. 14(10B):2121-2127;(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3) NP_000363.1;NM_000372.4; Bishop, D. T. et al (2009) Nat. Genet. 41 (8):920-925; Nan,H. et al (2009) Int. J. Cancer 125 (4):909-917;(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627)NP_001103373.1; NM_001109903.1; Clark, H. F. et al (2003) Genome Res. 13(10):2265-2270; Scherer, S. E. et al (2006) Nature 440 (7082):346-351(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;D15Ertd747e) NP_078807.1; NM_024531.3; Ericsson, T. A. et al (2003)Proc. Natl. Acad. Sci. U.S.A. 100 (11):6759-6764; Takeda, S. et al(2002) FEBS Lett. 520 (1-3):97-101.

The parent antibody may also be a fusion protein comprising analbumin-binding peptide (ABP) sequence (Dennis et al. (2002) “AlbuminBinding As A General Strategy For Improving The Pharmacokinetics OfProteins” J Biol Chem. 277:35035-35043; WO 01/45746). Antibodies of theinvention include fusion proteins with ABP sequences taught by: (i)Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV,page 35038; (ii) US 20040001827 at [0076]; and (iii) WO 01/45746 atpages 12-13, and all of which are incorporated herein by reference.

To prepare a cysteine engineered antibody by mutagenesis, DNA encodingan amino acid sequence variant of the starting polypeptide is preparedby a variety of methods known in the art. These methods include, but arenot limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.Variants of recombinant antibodies may be constructed also byrestriction fragment manipulation or by overlap extension PCR withsynthetic oligonucleotides. Mutagenic primers encode the cysteine codonreplacement(s). Standard mutagenesis techniques can be employed togenerate DNA encoding such mutant cysteine engineered antibodies.General guidance can be found in Sambrook et al Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel et al Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encodes an amino acid sequence comprising the VLand/or an amino acid sequence comprising the VH of the antibody (e.g.,the light and/or heavy chains of the antibody). In a further embodiment,one or more vectors (e.g., expression vectors) comprising such nucleicacid are provided. In a further embodiment, a host cell comprising suchnucleic acid is provided. In one such embodiment, a host cell comprises(e.g., has been transformed with): (1) a vector comprising a nucleicacid that encodes an amino acid sequence comprising the VL of theantibody and an amino acid sequence comprising the VH of the antibody,or (2) a first vector comprising a nucleic acid that encodes an aminoacid sequence comprising the VL of the antibody and a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VH of the antibody. In one embodiment, the host cell is eukaryotic,e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,Sp20 cell). In one embodiment, a method comprises culturing a host cellcomprising a nucleic acid encoding the antibody, as provided above,under conditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production, nucleic acid encoding an antibody, e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such nucleic acid maybe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Suitablehost cells for cloning or expression of antibody-encoding vectorsinclude prokaryotic or eukaryotic cells described herein. For example,antibodies may be produced in bacteria, in particular when glycosylationand Fc effector function are not needed. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos.5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006). Suitable host cells for theexpression of glycosylated antibody are also derived from multicellularorganisms (invertebrates and vertebrates). Examples of invertebratecells include plant and insect cells. Numerous baculoviral strains havebeen identified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts, such as those described in U.S.Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants). Vertebrate cells may also be used as hosts. Forexample, mammalian cell lines that are adapted to grow in suspension maybe useful. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line(293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol.36:59 (1977); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980);monkey kidney cells (CV1); African green monkey kidney cells (VERO-76);human cervical carcinoma cells (HELA); canine kidney cells (MDCK;buffalo rat liver cells (BRL 3A); human lung cells (W138); human livercells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68(1982); MRC 5 cells; and FS4 cells. Other useful mammalian host celllines include Chinese hamster ovary (CHO) cells, including DHFR-CHOcells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); andmyeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certainmammalian host cell lines suitable for antibody production, see, e.g.,Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed.,Humana Press, Totowa, N.J.), pp. 255-268 (2003).

Site-directed mutagenesis is one method for preparing substitutionvariants, i.e. mutant proteins (Carter (1985) et al Nucleic Acids Res.13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59; and Kunkel et al(1987) Proc. Natl. Acad. Sci. USA 82:488). Starting DNA is altered byfirst hybridizing an oligonucleotide encoding the desired mutation to asingle strand of such starting DNA. After hybridization, a DNApolymerase is used to synthesize an entire second strand, using thehybridized oligonucleotide as a primer, and using the single strand ofthe starting DNA as a template. Thus, the oligonucleotide encoding thedesired mutation is incorporated in the resulting double-stranded DNA.Site-directed mutagenesis may be carried out within the gene expressingthe protein to be mutagenized in an expression plasmid and the resultingplasmid may be sequenced to confirm the introduction of the desiredcysteine replacement mutations (Liu et al (1998) J. Biol. Chem.273:20252-20260). Site-directed mutagenesis protocols and formats arewidely available, e.g. QuikChange® Multi Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.).

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide. See Higuchi, (1990) in PCR Protocols, pp.177-183, Academic Press; Ito et al (1991) Gene 102:67-70; Bernhard et al(1994) Bioconjugate Chem., 5:126-132; and Vallette et al (1989) Nuc.Acids Res., 17:723-733. Briefly, when small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al (1985) Gene, 34:315-323. Thestarting material is the plasmid (or other vector) comprising thestarting polypeptide DNA to be mutated. The codon(s) in the starting DNAto be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using the abovedescribed oligonucleotide-mediated mutagenesis method to introduce themat appropriate locations in the starting polypeptide DNA. The plasmidDNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence. Mutant DNA containing the encoded cysteine replacementscan be confirmed by DNA sequencing.

Single mutations are also generated by oligonucleotide directedmutagenesis using double stranded plasmid DNA as template by PCR basedmutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A LaboratoryManual, 3rd edition; Zoller et al (1983) Methods Enzymol. 100:468-500;Zoller, M. J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500).

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability.

CBI Dimer Drug Moiety

An antibody-drug conjugate compound of the invention comprises a CBIdimer drug moiety D. A CBI dimer drug moiety may be comprised of two CBIdrug units or one CBI drug unit and one PBD drug unit.

The CBI dimer drug moiety D has the formula:

where

R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b);

R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b);

R^(a) and R^(b) are independently selected from H and C₁-C₆ alkyloptionally substituted with one or more F,

or R^(a) and R^(b) form a five or six membered heterocyclyl group;

T is a tether group selected from C₃-C₁₂ alkylene, Y, (C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₁-C₆ alkylene)-Y—(C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₂-C₆ alkenylene)-Y—(C₂-C₆ alkenylene),and (C₂-C₆ alkynylene)-Y—(C₂-C₆ alkynylene);

where Y is independently selected from O, S, NR¹, aryl, and heteroaryl;

where alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃, N(CH₃)₂,OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally substituted withone or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with a bond to L;

D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

X¹ and X² are independently selected from O and NR³, where R³ isselected from H and C₁-C₆ alkyl optionally substituted with one or moreF;

R⁴ is H, CO₂R, where R is C₁-C₆ alkyl or benzyl; and

R⁵ is H or C₁-C₆ alkyl.

In certain embodiments, Y is phenyl, pyridyl,1-methyl-1H-benzo[d]imidazole, or [1,2,4]triazolo[1,5-a]pyridine.

CBI dimer drug moiety D compounds include those in Table 1 which areuseful for preparing Linker-CBI drug intermediates of Table 3.

TABLE 1 CBI dimer drug moiety compounds No. Structure Name MW 51b

1,5-bis((S)-1-(chloromethyl)- 5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione 563.47 53j

1-((S)-5-amino-1- (chloromethyl)-1H- benzo[e]indol-3(2H)-yl)-5-((S)-1-(chloromethyl)-5- hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione 562.49 53p

1-((R)-5-amino-1- (chloromethyl)-1H- benzo[e]indol-3(2H)-yl)-5-((R)-1-(chloromethyl)-5- hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione 562.49 11

  (racemic) 2,2′-azanediylbis(1-(1- (chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)- yl)ethanone)hydrochloride 600.92 12

  (racemic) 1,5-bis(1-(chloromethyl)-5- hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione 563.47 13

  (racemic) 1,7-bis(1-(chloromethyl)-5- hydroxy-1H-benzo[e]indol-3(2H)-yl)heptane-1,7-dione 591.52 14

(S)-8-(6-((S)-1- (chloromethyl)-5-hydroxy- 1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-7-methoxy- 2,3-dihydro-1H- benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one 576.08 15

(S)-(1-methyl-1H-pyrrole- 2,5-diyl)bis(((S)-1- (chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)- yl)methanone) 622.13 16

N-(2,5-bis((E)-3-((S)-1- (chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)- 3-oxoprop-1- enyl)phenyl)acetamide 728.17 17

(S,2E,2′E)-3,3′-(2-methoxy- 1,4-phenylene)bis(1-((S)-1-(chloromethyl)-5-hydroxy- 1H-benzo[e]indol-3(2H)- yl)prop-2-en-1-one)701.16 18

(S,2E,2′E)-3,3′-(1-methyl- 1H-pyrrole-2,5-diyl)bis(1-((S)-1-(chloromethyl)-5- hydroxy-1H-benzo[e]indol-3(2H)-yl)prop-2-en-1-one) 650.16 19

(S)-3,3′-(2-methoxy-1,4- phenylene)bis(1-((S)-1-(chloromethyl)-5-hydroxy- 1H-benzo[e]indol-3(2H)- yl)prop-2-yn-1-one)697.13

Tether reagents useful for preparing CBI dimer drug moiety D compoundsinclude those in Table 2

TABLE 2 Tether reagents No. Structure Name MW 21

(2E,2′E)-3,3′-(2-(di-tert- butoxyphosphoryloxy)-3-(3-(6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1- yl)hexanamido)propanamido)-1,4-phenylene)diacrylic acid 705.69 22

(2E,2′E)-3,3′-(2-(2-(6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)ethyl)-1- methyl-1H- benzo[d]imidazole-4,7-diyl)diacrylic acid 508.52 23

(2E,2′E)-3,3′-(2-(2-(6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)ethyl)- [1,2,4]triazolo[1,5-a]pyridine-5,8-diyl)diacrylic acid 495.48 24

(2E,2′E)-3,3′-(6-(3-(6-(2,5- dioxo-2,5-dihydro-1H-pyrrol- 1-yl)hexanamido)propanamido) pyridine-2,5-diyl)diacrylic acid 498.49 25

(2E,2′E)-3,3′-(2-(2-(2-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)-1,4- phenylene)diacrylic acid 401.37 26

(2E,2′E)-3,3′-(2-(2-(2,5- dioxo-2,5-dihydro-1H-pyrrol- 1-yl)ethoxy)-1,4-phenylene)diacrylic acid 357.08 27

2-(3-(6-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1- yl)hexanamido)propanamido)terephthalic acid 445.42 28

3,3′-(2-(3-(6-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)- 1,4-phenylene)dipropiolic acid 493.47Linkers

An antibody-drug conjugate (ADC) compound of the invention comprises alinker L having the formula:-Str-(Pep)_(m)-(Sp)_(n)-

where Str is a stretcher unit covalently attached to the antibody; Pepis an optional peptide unit of two to twelve amino acid residues, Sp isan optional spacer unit covalently attached to a dimer drug moiety, andm and n are independently selected from 0 and 1.

In an exemplary embodiment, Str has the 1,3-disubstituted,pyrrolidine-2,5-dione (succinimide) formula:

wherein R⁶ is selected from the group consisting of C₁-C₁₀ alkylene,C₃-C₈ carbocyclyl, O—(C₁-C₈ alkyl), arylene, C₁-C₁₀ alkylene-arylene,arylene-C₁-C₁₀ alkylene, C₁-C₁₀ alkylene-(C₃-C₈ carbocyclyl), (C₃-C₈carbocyclyl)-C₁-C₁₀ alkylene, C₃-C₈ heterocyclyl, C₁-C₁₀ alkylene-(C₃-C₈heterocyclyl), (C₃-C₈ heterocyclyl)-C₁-C₁₀ alkylene, C₁-C₁₀alkylene-C(O)N(R⁸)—C₂-C₆ alkylene-N(R⁸), N(R⁸)—(C₂-C₆ alkylene), and(CH₂CH₂O)_(r)—CH₂; where R⁸ is H or C₁-C₆ alkyl, and r is an integerranging from 1 to 10.

The 1,3-disubstituted, pyrrolidine-2,5-dione embodiments of the Strstretcher unit may be formed from conjugation of cysteine thiols ofantibodies with the maleimide group of linker-drug intermediates, suchas those in Table 4.

In another exemplary embodiment, Str has the formula:

wherein R⁷ is selected from C₁-C₁₀ alkylene, C₁-C₁₀ alkylene-O,N(R⁸)—(C₂-C₆ alkylene)-N(R⁸), N(R⁸)—(C₂-C₆ alkylene), and(CH₂CH₂O)_(r)—CH₂; where R⁸ is H or C₁-C₆ alkyl, and r is an integerranging from 1 to 10.

In another exemplary embodiment, Str has the formula:

wherein R⁹ is selected from C₁-C₁₀ alkylene, C₁-C₁₀ alkylene-O, (C₂-C₆alkylene)-N(R⁸), and (CH₂CH₂O)_(r)—CH₂; where R⁸ is H or C₁-C₆ alkyl,and r is an integer ranging from 1 to 10.

In another exemplary embodiment, L forms a disulfide bond with acysteine amino acid of the antibody, and R⁹ is C₂-C₆ alkylene-O wherealkylene is optionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂,NHCH₃, N(CH₃)₂, OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionallysubstituted with one or more F.

Linker reagents and linker-drug intermediates may have a peptide unit oftwo to twelve or more amino acid residues.

In an exemplary embodiment, m is 1 and Pep comprises two to twelve aminoacid residues independently selected from glycine, alanine,phenylalanine, lysine, arginine, valine, and citrulline.

In an exemplary embodiment, Pep is valine-citrulline.

Peptide-linker reagents were prepared as described (WO 2012113847; U.S.Pat. Nos. 7,659,241; 7,498,298; US 2009/0111756; US 2009/0018086; U.S.Pat. No. 6,214,345; Dubowchik et al (2002) Bioconjugate Chem.13(4):855-869).

In an exemplary embodiment, Sp comprises para-aminobenzyl orpara-aminobenzyloxycarbonyl.

Table 3 shows exemplary Linker reagents useful for preparing Linker-CBIdrug intermediates of Table 4.

TABLE 3 Peptide-linker reagents No. Structure Name 41

6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-N-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)- 1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan- 2-yl)hexanamide 42

N-((S)-1-((S)-1-(4- (chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)- 3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 43

4-((S)-2-((S)-2-(6-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5- ureidopentanamido)benzyl 4-nitrophenyl carbonateLinker-Drug Intermediates Useful for ADC

Linker-drug intermediates useful for conjugation to antibodies toprepare antibody-drug conjugates have the formula:X-L-D

wherein:

X is a reactive functional group selected from maleimide, thiol, amino,bromide, bromoacetamido, iodoacetamido, p-toluenesulfonate, iodide,hydroxyl, carboxyl, pyridyl disulfide, and N-hydroxysuccinimide;

L is a linker having the formula:-Str-(Pep)_(m)-(Sp)_(n)-

where Str is a stretcher unit covalently attached to reactive functionalgroup X; Pep is an optional peptide unit of two to twelve amino acidresidues, Sp is an optional spacer unit covalently attached to a dimerdrug moiety, and m and n are independently selected from 0 and 1;

D is the dimer drug moiety having the formula:

where

R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R^(a) and R^(b) are independently selected from H and C₁-C₆ alkyloptionally substituted with one or more F, or R^(a) and R^(b) form afive or six membered heterocyclyl group;

T is a tether group selected from C₃-C₁₂ alkylene, Y, (C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₁-C₆ alkylene)-Y—(C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₂-C₆ alkenylene)-Y—(C₂-C₆ alkenylene),and (C₂-C₆ alkynylene)-Y—(C₂-C₆ alkynylene);

where Y is independently selected from O, S, NR¹, aryl, and heteroaryl;

where alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃, N(CH₃)₂,OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally substituted withone or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with a bond to L;

D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

X¹ and X² are independently selected from O and NR³, where R³ isselected from H and C₁-C₆ alkyl optionally substituted with one or moreF;

R⁴ is H, CO₂R, or a bond to L, where R is C₁-C₆ alkyl or benzyl; and

R⁵ is H or C₁-C₆ alkyl.

Linker-drug intermediates of Table 4 were prepared by coupling a CBIdimer drug moiety with a linker reagent, as exemplified in Examples 1-18and FIGS. 1-24.

TABLE 4 Linker-CBI drug intermediates No. Structure Name 51

(S)-1- (chloromethyl)- 3-(5-((S)-1- (chloromethyl)-5- hydroxy-1H-benzo[e]indol- 3(2H)-yl)-5- oxopentanoyl)- 2,3-dihydro-1H-benzo[e]indol- 5-yl 2-(2-bromo-N- methylacetamido) ethyl(methyl)carbamate 52

(11S,11aS)-tert- butyl 8-(6-((S)-1- (chloromethyl)- 5-(4-((S)-2-((S)-2-(6-(2,5-dioxo- 2,5-dihydro-1H- pyrrol-1- yl)hexanamido)-3-methylbutanamido)-5- ureidopentanamido) benzyloxy)- 1H-benzo[e]indol-3(2H)-yl)-6- oxohexyloxy)- 11-hydroxy-7- methoxy-5- oxo-2,3,11,11a-tetrahydro-1H- benzo[e]pyrrolo[1,2- a][1,4]diazepine- 10(5H)-carboxylate53

N-((R)-1- (chloromethyl)-3- (5-((R)-1- (chloromethyl)- 5-hydroxy-1H-benzo[e]indol- 3(2H)-yl)-5- oxopentanoyl)-2,3- dihydro-1H-benzo[e]indol-5- yl)-6-(2,5-dioxo- 2,5-dihydro-1H- pyrrol-1-yl)hexanamide 54

N-(4-(((S)-1- (chloromethyl)- 3-(6-((S)-7- methoxy-5-oxo- 2,3,5,11a-tetrahydro-1H- benzo[e]pyrrolo [1,2-a][1,4] diazepin-8- yloxy)hexanoyl)-2,3-dihydro-1H- benzo[e]indol-5- yloxy)methyl) phenyl)-6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1- yl)hexanamide 55

N-((S)-1-((S)- 1-(4-(((S)-1- (chloromethyl)- 3-(5-((S)-1-(chloromethyl)- 5-hydroxy-1H- benzo[e]indol- 3(2H)-yl)-5- oxopentanoyl)-2,3-dihydro-1H- benzo[e]indol-5- yloxy)methyl) phenylamino)-1- oxo-5-ureidopentan-2- ylamino)- 3-methyl-1- oxobutan-2-yl)- 6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1- yl)hexanamide 56

N-((S)-1-((S)- 1-(4-(((S)-1- (chloromethyl)- 3-(6-((S)-7- methoxy-5-oxo-2,3,5,11a- tetrahydro-1H- benzo[e]pyrrolo [1,2-a][1,4] diazepin-8-yloxy)hexanoyl)- 2,3-dihydro-1H- benzo[e]indol-5- yloxy)methyl)phenylamino)-1- oxo-5- ureidopentan- 2-ylamino)- 3-methyl-1-oxobutan-2-yl)-6- (2,5-dioxo-2,5- dihydro-1H- pyrrol-1- yl)hexanamide 57

(S)-1- (chloromethyl)- 3-(5-((S)-1- (chloromethyl)-5- (phosphonooxy)-1H-benzo[e]indol- 3(2H)-yl)-5- oxopentanoyl)- 2,3-dihydro-1H-benzo[e]indol-5- yl 2-(6-(2,5- dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-N-methylhexanamido) ethyl(methyl) carbamate 58

(S)-1- (chloromethyl)- 3-(5-((S)-1- (chloromethyl)- 5-(4-((S)-2-((S)-2-(6-(2,5-dioxo- 2,5-dihydro-1H- pyrrol-1- yl)hexanamido)-3-methylbutanamido)- 5- ureidopentanamido) benzyloxy)- 1H-benzo[e]indol-3(2H)-yl)-5- oxopentanoyl)-2,3- dihydro-1H- benzo[e]indol-5- yl 4-methylpiperazine- 1-carboxylate 59

(S)-1- (chloromethyl)- 3-(5-((S)-1- (chloromethyl)- 5-(4-((S)-2-((S)-2-(6-(2,5-dioxo- 2,5-dihydro-1H- pyrrol-1- yl)hexanamido)-3-methylbutanamido)- 5- ureidopentanamido) benzyloxy)- 1H-benzo[e]indol-3(2H)-yl)-5- oxopentanoyl)- 2,3-dihydro-1H- benzo[e]indol- 5-yldihydrogen phosphate 60

N-((S)-1-((S)- 1-(4-(((S)-1- (chloromethyl)- 3-(5-((S)-1-(chloromethyl)- 5-hydroxy-1H- benzo[e]indol- 3(2H)-yl)-5- oxopentanoyl)-2,3-dihydro-1H- benzo[e]indol-5- yloxy)methyl) phenylamino)-1- oxo-5-ureidopentan- 2-ylamino)- 3-methyl-1- oxobutan-2-yl)- 6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1- yl)hexanamide 61

2-(pyridin-2- yldisulfanyl)ethyl (S)-1- (chloromethyl)- 3-(5-((S)-1-(chloromethyl)-5- (phosphonooxy)- 1H-benzo[e]indol- 3(2H)-yl)-5-oxopentanoyl)- 2,3-dihydro-1H- benzo[e]indol- 5-ylcarbamate 62

2-(pyridin-2- yldisulfanyl) propyl (S)-1- (chloromethyl)- 3-(5-((S)-1-(chloromethyl)-5- (phosphonooxy)- 1H-benzo[e]indol- 3(2H)-yl)-5-oxopentanoyl)- 2,3-dihydro-1H- benzo[e]indol- 5-ylcarbamate 63

(S)-1- (chloromethyl)- 3-(5-((S)-1- (chloromethyl)- 5-hydroxy-1H-benzo[e]indol- 3(2H)-yl)-5- oxopentanoyl)- 2,3-dihydro-1H-benzo[e]indol- 5-yl 2-(6-(2,5- dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-N-methylhexanamido) ethyl(methyl) carbamate 64

2-(pyridin-2- yldisulfanyl) ethyl (S)-1- (chloromethyl)- 3-(5-((S)-1-(chloromethyl)- 5-hydroxy-1H- benzo[e]indol- 3(2H)-yl)-5- oxopentanoyl)-2,3-dihydro-1H- benzo[e]indol- 5-ylcarbamate 65

(11aS)-4-((S)- 6-amino-2-((S)-2- (6-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1- yl)hexanamido)-3- methylbutanamido) hexanamido) benzyl8-(6-((S)- 1-(chloromethyl)- 5-(4- methylpiperazine- 1-carbonyloxy)-1H-benzo[e]indol- 3(2H)-yl)-6- oxohexyloxy)-11- hydroxy-7- methoxy-5-oxo-2,3,11,11a- tetrahydro-1H- benzo[e]pyrrolo [1,2-a][1,4]diazepine-10(5H)- carboxylate 66

N-(3-(2,5-bis((E)- 3-((S)-1- (chloromethyl)- 5-phosphonoxy-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl) phenylamino)-3-oxopropyl)-6- (2,5-dioxo-2,5- dihydro-1H-pyrrol- 1-yl)hexanamide 67

N-(3-(2,5-bis((E)- 3-((S)-1- (chloromethyl)-5- hydroxy-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl) phenylamino)-3-oxopropyl)-6- (2,5-dioxo-2,5- dihydro-1H-pyrrol- 1-yl)hexanamide 68

(S)-1- (chloromethyl)- 3-((E)-3-(4- ((E)-3-((S)-1- (chloromethyl)-5-hydroxy-1H- benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl)- 2-(3-(6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1- yl)hexanamido) propanamido)phenyl)acryloyl)- 2,3-dihydro-1H- benzo[e]indol- 5-yl dihydrogenphosphate 69

N-(3-(2,5- bis((E)-3-((S)-1- (chloromethyl)- 5-(4- methylpiperazine-1-carbonyloxy)- 1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl)phenylamino)- 3-oxopropyl)- 6-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl) hexanamide 70

N-(3-(2,5-bis((E)- 3-((S)-1- (chloromethyl)-5- ((2S,3S,4S,5R,6S)-3,4,5-trihydroxy- tetrahydro-2H- pyran-2-carboxyl- 6-oxy)-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl) phenylamino)-3-oxopropyl)-6- (2,5-dioxo-2,5- dihydro-1H-pyrrol- 1-yl)hexanamide 71

N-(3-(2,5-bis((E)- 3-((S)-1- (chloromethyl)- 5-phosphonoxy-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl) phenylamino)-6-(2,5-dioxo-2,5- dihydro-1H- pyrrol-1-yl)-N- methyl-N-(3- (methylamino)-3-oxopropyl) hexanamide 72

2-(pyridin-2- yldisulfanyl) propyl2,5-bis((E)- 3-((S)-1-(chloromethyl)-5- (phosphonooxy)- 1H-benzo[e]indol- 3(2H)-yl)-3-oxoprop-1-enyl) phenylcarbamate 73

N-(3-(2,5- bis((E)-3-((S)-1- (chloromethyl)- 5-phosphonoxy-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl) phenylamino)-7-(2-bromo-N- methylacetamido)- N-(3-oxopropyl) heptanamide 74

N-(2-(2,5- bis((S)-1- (chloromethyl)- 5-phosphonoxy- 2,3-dihydro-1H-benzo[e]indole- 3-carbonyl)-1H- pyrrol-1- yl)ethyl)-6- (2,5-dioxo-2,5-dihydro-1H- pyrrol-1- yl)hexanamide 75

N-(2-(2,5- bis((E)-3-((S)-1- (chloromethyl)- 5-phosphonoxy-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1- enyl)-1H-pyrrol-1-yl)ethyl)-6- (2,5-dioxo-2,5- dihydro-1H-pyrrol- 1-yl)hexanamide 76

N-(3-(2,5- bis((E)-3-((S)-1- (chloromethyl)- 5-phosphonoxy-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl) phenylamino,2-phosphonoxy)- 3-oxopropyl)-6- (2,5-dioxo-2,5- dihydro-1H- pyrrol-1-yl)hexanamide 77

N-(3-(2,5- bis((E)-3-((S)-1- (chloromethyl)- 5-phosphonoxy-1H-benzo[e]indol- 3(2H)-yl)-3- oxoprop-1-enyl)- 1-methyl-1H-benzo[d]imidazol- 2-yl)ethyl)-6- (2,5-dioxo-2,5- dihydro-1H- pyrrol-1-yl)hexanamide 78

[(1S)-1- (chloromethyl)- 3-[(E)-3- [4-[(E)-3-[(1S)- 1-(chloromethyl)-5-phosphonooxy- 1,2-dihydrobenzo[e] indol-3-yl]-3- oxo-prop-1-enyl]-2-[2-[2-(2,5- dioxopyrrol-1- yl)ethoxy]ethoxy] phenyl]prop-2-enoyl]-1,2- dihydrobenzo[e] indol-5-yl] dihydrogen phosphate 79

[(1S)-1- (chloromethyl)- 3-[(E)-3- [4-[(E)-3-[(1S)- 1-(chloromethyl)-5-phosphonooxy- 1,2-dihydrobenzo [e]indol-3-yl]-3- oxo-prop-1-enyl]-2-[2-(2,5- dioxopyrrol-1- yl)ethoxy]phenyl] prop-2-enoyl]-1,2-dihydrobenzo[e] indol-5-yl] dihydrogen phosphate 80

2-(2- pyridyldisulfanyl) propyl N- [1-(chloromethyl)- 3-[5-[1-(chloromethyl)- 5-hydroxy-1,2- dihydrobenzo[e] indol-3-yl]-5-oxo-pentanoyl]- 1,2-dihydrobenzo [e]indol-5- yl]carbamate 81

2-(2- pyridyldisulfanyl) propyl 3-[6-[1- (chloromethyl)- 5-(4-methylpiperazine- 1-carbonyl)oxy- 1,2-dihydrobenzo [e]indol-3-yl]-6-oxo-hexoxy]-6- hydroxy-2- methoxy-11- oxo-6a,7,8,9- tetrahydro-6H-pyrrolo[2,1-c][1,4] benzodiazepine- 5-carboxylate 82

2-(2- pyridyldisulfanyl) propyl 3-[6-[1- (chloromethyl)-5- phosphonooxy-1,2-dihydrobenzo [e]indol-3-yl]- 6-oxo-hexoxy]- 6-hydroxy-2- methoxy-11-oxo-6a,7,8,9- tetrahydro-6H- pyrrolo[2,1-c][1,4] benzodiazepine-5-carboxylate 83

2-(2- pyridyldisulfanyl) propyl 3-[6-[1- (chloromethyl)- 5-hydroxy-1,2-dihydrobenzo[e] indol-3-yl]-6- oxo-hexoxy]- 6-hydroxy-2- methoxy-11-oxo-6a,7,8,9- tetrahydro-6H- pyrrolo[2,1-c][1,4] benzodiazepine-5-carboxylate 84

(1S)-1- (chloromethyl)- 3-((2E)-3-{4- ((1E)-3-{(1S)-1- (chloromethyl)-5-[(6-methyl-β-D- gluco- pyranuronosyl) oxy]-1,2- dihydro-3H-benzo[e]indol- 3-yl}-3-oxo-1- propenyl)-2-[(3- {[6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl) hexanoyl]amino} propanoyl)amino] phenyl}-2-propenoyl)-1,2- dihydro-3H- benzo[e]indol- 5-yl methyl β-D- gluco-pyranosiduronateAntibody-Drug Conjugates (ADC)

The antibody-drug conjugate (ADC) compounds of the invention comprise anantibody specific for a tumor-associated antigen linked to a potent CBIdimer drug moiety, and include those with therapeutic activity,effective against a number of hyperproliferative disorders, includingcancer. The biological activity of the drug moiety is modulated byconjugation to an antibody. The ADC of the invention selectively deliveran effective dose of the CBI dimer drug, or toxin, to tumor cell or sitewhereby greater selectivity, i.e. a lower efficacious dose, may beachieved while increasing the therapeutic index (“therapeutic window”).In an exemplary embodiment, the ADC compounds include acysteine-engineered antibody conjugated, i.e. covalently attached by alinker, to the CBI dimer drug moiety.

An antibody-drug conjugate compound of the invention has the formula:Ab-(L-D)_(p)

wherein:

Ab is an antibody;

L is a linker having the formula:-Str-(Pep)_(m)-(Sp)_(n)-

where Str is a stretcher unit covalently attached to the antibody; Pepis an optional peptide unit of two to twelve amino acid residues, Sp isan optional spacer unit covalently attached to a dimer drug moiety, andm and n are independently selected from 0 and 1;

p is an integer from 1 to 8;

D is the dimer drug moiety having the formula:

where

R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;

R^(a) and R^(b) are independently selected from H and C₁-C₆ alkyloptionally substituted with one or more F,

or R^(a) and R^(b) form a five or six membered heterocyclyl group;

T is a tether group selected from C₃-C₁₂ alkylene, Y, (C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₁-C₆ alkylene)-Y—(C₁-C₆alkylene)-Y—(C₁-C₆ alkylene), (C₂-C₆ alkenylene)-Y—(C₂-C₆ alkenylene),and (C₂-C₆ alkynylene)-Y—(C₂-C₆ alkynylene);

where Y is independently selected from O, S, NR¹, aryl, and heteroaryl;

where alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃, N(CH₃)₂,OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally substituted withone or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently andoptionally substituted with a bond to L;

D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

X¹ and X² are independently selected from O and NR³, where R³ isselected from H and C₁-C₆ alkyl optionally substituted with one or moreF;

R⁴ is H, CO₂R where R is C₁-C₆ alkyl or benzyl; and

R⁵ is H.

In one embodiment, R^(a) and R^(b) form a five or six memberedheterocyclyl group selected from N-methylpiperazinyl, morpholinyl,piperidyl, and pyrrolidinyl.

In one embodiment, the drug moiety may is joined to the antibody via aprotease cleavable, peptide linker cleavable by cathepsin B, a lysosomalprotease found in most mammalian cell types (U.S. Pat. No. 6,214,345;Dubowchik et al (2002) Bioconj. Chem. 13:855-869). While the inventionis not limited or defined by any particular mechanism of action, the ADCmay act as a pro-drug in that the drug is inactive until the linker iscleaved. The ADC is able to concentrate the active drug specifically ina tumor cell location where disease may be poorly treated byconventional chemotherapy. In other embodiments, the drug moiety isattached to the antibody via a non-peptide, non-protease cleavablelinker, which may include functionality such as disulfide orsuccinimidyl groups.

The number of drug moieties which may be conjugated via a reactivelinker moiety to an antibody molecule may be limited by the number offree cysteine residues, which are introduced by the methods describedherein. Exemplary ADC therefore comprise antibodies which have 1, 2, 3,or 4 engineered cysteine amino acids (Lyon, R. et al (2012) Methods inEnzym. 502:123-138).

In one embodiment, the antibody-drug conjugate compound has the formula:

where AA1 and AA2 are independently selected from an amino acid sidechain. The amino acid side chain is independently selected from H, —CH₃,—CH₂(C₆Hs), —CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂NHC(NH)NH₂, —CHCH(CH₃)CH₃, and—CH₂CH₂CH₂NHC(O)NH₂.

In one embodiment, the antibody-drug conjugate compound has the formula:

In one embodiment, the antibody-drug conjugate compound has the formula:

In one embodiment, the antibody-drug conjugate compound has the formula:

In one embodiment, the antibody-drug conjugate compound has the formula:

In one embodiment, the antibody-drug conjugate compound has the formula:

In one embodiment, the antibody-drug conjugate compound has the formula:

In one embodiment, the antibody-drug conjugate compound has the formula:

In one embodiment, the antibody-drug conjugate compound has the formula:

where R⁷ is independently selected from H and C₁-C₁₂ alkyl.

In one embodiment, p is 1, 2, 3 or 4.

In one embodiment, p is 2.

In one embodiment, D is a moiety selected from the compounds listed inTable 1 or a derivative thereof.

In one embodiment, L-D is a moiety selected from the compounds listed inTable 4 or a derivative thereof.

In one embodiment, the present invention relates to a conjugate selectedfrom the molecules listed in Table 5.

Drug Loading of ADC

The drug loading is the average number of CBI drug moieties perantibody. Drug loading may range from 1 to 8 drugs (D) per antibody(Ab), i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalentlyattached to the antibody. Compositions of ADC include collections ofantibodies conjugated with a range of drugs, from 1 to 8. The averagenumber of drugs per antibody in preparations of ADC from conjugationreactions may be characterized by conventional means such as massspectroscopy, ELISA assay, electrophoresis, and HPLC. The quantitativedistribution of ADC in terms of p may also be determined. By ELISA, theaveraged value of p in a particular preparation of ADC may be determined(Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al(2005) Clin. Cancer Res. 11:843-852). However, the distribution of p(drug) values is not discernible by the antibody-antigen binding anddetection limitation of ELISA. Also, ELISA assay for detection ofantibody-drug conjugates does not determine where the drug moieties areattached to the antibody, such as the heavy chain or light chainfragments, or the particular amino acid residues. In some instances,separation, purification, and characterization of homogeneous ADC wherep is a certain value from ADC with other drug loadings may be achievedby means such as reverse phase HPLC or electrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading, e.g. p>5, may cause aggregation,insolubility, toxicity, or loss of cellular permeability of certainantibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties isconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with thelinker-drug intermediate (X-L-D) or linker reagent. Only the mostreactive lysine groups may react with an amine-reactive linker reagent.Also, only the most reactive cysteine thiol groups may react with athiol-reactive linker reagent or linker-drug intermediate. Generally,antibodies do not contain many, if any, free and reactive cysteine thiolgroups which may be linked to a drug moiety. Most cysteine thiolresidues in the antibodies of the compounds exist as disulfide bridgesand must be reduced with a reducing agent such as dithiothreitol (DTT)or TCEP, under partial or total reducing conditions. The loading(drug/antibody ratio, “DAR”) of an ADC may be controlled in severaldifferent manners, including: (i) limiting the molar excess oflinker-drug intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a linker-drug intermediate, or linker reagent followed byCBI dimer drug moiety reagent, then the resulting product is a mixtureof ADC compounds with a distribution of drug moieties attached to anantibody, e.g. 1, 2, 3, etc. Liquid chromatography methods such aspolymeric reverse phase (PLRP) and hydrophobic interaction (HIC) mayseparate compounds in the mixture by drug loading value. Preparations ofADC with a single drug loading value (p) may be isolated, however, thesesingle loading value ADCs may still be heterogeneous mixtures becausethe drug moieties may be attached, via the linker, at different sites onthe antibody. Thus the antibody-drug conjugate compositions of theinvention include mixtures of antibody-drug conjugate compounds wherethe antibody has one or more drug moieties and where the drug moietiesmay be attached to the antibody at various amino acid residues.

Methods of Preparing Antibody-Drug Conjugates

Exemplary antibody-drug conjugates (ADC) compounds 101-139 of theinvention were prepared from linker-drug intermediates 51-68 accordingto Example 20 and as shown in Table 4.

TABLE 5 Antibody-drug conjugates (ADC) linker-CBI interme- diate No. ADCformula (Table 4) DAR* 101 Thio hu anti-CD22 HC A121C-MC-vc-PAB- 55 1.7(CBI dimer) 102 Thio hu anti-Her2 HC A121C-MC-vc-PAB- 55 1.8 (CBI dimer)103 Thio Hu Anti-Her2 4D5 HC A118C-MC-vc- 56 1.6 PAB-(CBI-PBD) 104 ThioHu Anti-CD22 10F4v3 HC A118C- 56 1.6 MC-vc-PAB-(CBI-PBD) 105 Thio HuAnti-Her2 4D5 HC A118C-MC-vc- 58 1.7 PAB-(CBI dimer MePip) 106 Thio HuAnti-Her2 4D5 HC A118C-MC- 57 2.0 MMED-(CBI dimer phos) 107 Thio HuAnti-Her2 4D5 HC A118C-MC-vc- 58 1.6 PAB-(CBI dimer MePip) 108 Thio HuAnti-CD22 10F4v3 HC A118C- 58 1.8 MC-vc-PAB-(CBI dimer MePip) 109 ThioHu Anti-Her2 4D5 HC A118C-MC- 57 1.9 MMED-(CBI dimer phos) 110 Thio HuAnti-CD22 10F4v3 HC A118C- 57 1.9 MC-MMED-(CBI dimer phos) 111 Thio HuAnti-CD22 10F4v3 HC A118C- 59 1.8 MC-vc-PAB-(CBI dimer phos) 112 Thio HuAnti-Her2 4D5 HC A118C-MC-vc- 59 1.9 PAB-(CBI dimer phos) 113 Thio HuAnti-Her2 4D5 HC A118C-DSE- 61 2.0 (CBI dimer phos) 114 Thio HuAnti-Her2 4D5 HC A118C-MC- 57 1.9 MMED-(CBI dimer phos) Phosphatasetreated 115 Thio Hu Anti-CD22 10F4v3 HC A118C- 63 1.9 MC-MMED-(CBIdimer) Phosphatase treated 116 Thio Hu Anti-CD22 10F4v3 HC A118C- 60 1.8MC-vc-PAB-(CBI dimer) Phosphatase treated 117 Thio Hu Anti-Her2 4D5 HCA118C-MC-vc- 60 1.9 PAB-(CBI dimer) Phosphatase treated 118 Thio HuAnti-Her2 4D5 HC A118C-DSE- 64 2.0 (CBI dimer) Phosphatase treated 119Thio Hu Anti-Her2 4D5 HC A118C-DSE- 61 1.9 (CBI dimer phos) 120 Thio HuAnti-CD22 10F4v3 HC A118C- 61 1.8 DSE-(CBI dimer phos) 121 Thio HuAnti-Her2 4D5 HC A118C-DSP- 62 1.8 (CBI dimer phos) 122 Thio HuAnti-CD22 10F4v3 HC A118C- 62 1.6 DSE-(CBI dimer phos) 123 Thio HuAnti-Her2 4D5 HC A118C-MC-vc- 65 1.9 PAB-(N10, PBD-CBI MePip) 124 ThioHu Anti-CD22 10F4v3 HC A118C- 65 1.7 MC-vc-PAB-(N10,PBD-CBI MePip) 125Thio Hu Anti-CD33 15G15.33 HC A118C- 57 1.6 MC-MMED-(CBI dimer phos) 126Thio Hu Anti-MUC16 3A5 HC A118C-MC- 66 1.8 ED-(CBI dimer DVB diphos) 127Thio Hu Anti-CD33 15G15.33 HC A118C- 66 1.5 MC-ED-(CBI dimer DVB diphos)128 Thio Hu Anti-MUC16 3A5 HC A118C-MC- 57 1.95 MMED-(CBI dimer phos)129 Thio Hu Anti-CD33 15G15.33 HC A118C- 68 2.0 MC-ED-(CBI dimer DVBphos) 130 Thio Hu Anti-MUC16 3A5 HC A118C-MC- 68 2.0 ED-(CBI dimer DVBphos) 131 Thio Hu Anti-CD33 15G15.33 HC A118C- 62 1.55 DSP-(CBI dimerphos) 132 Thio Hu Anti-MUC16 3A5 HC A118C- 62 DSP-(CBI dimer phos) 133Thio Hu Anti-CD33 15G15.33 LC V205C- 62 1.9 DSP-(CBI dimer phos) 134Thio Hu Anti-NaPi2b 10H1.11.4B HC 81 1.8 A118C-(Compound 81) 135 Thio HuAnti-NaPi2b 10H1.11.4B HC 78 1.8 A118C-(Compound 78) 136 Thio HuAnti-NaPi2b 10H1.11.4B HC 72 2 A118C-(Compound 72) 137 Thio HC anti-CD33(GM15.33)-(Compound 81 1.8 81) 138 Thio Hu Anti-CD33 (GM15.33)-(Compound78 1.9 78) 139 Thio Hu Anti-CD33 (GM15.33)-(Compound 72 1.57 72) *DAR =drug/antibody ratio average ** A118C (EU numbering) = A121C (Sequentialnumbering) = A114C (Kabat numbering)Anti-NaPi2b Antibodies

The anti-NaPi2b antibodies of ADC in Table 5 comprise three light chainhypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavy chainhypervariable regions (HVR-H1, HVR-H2 and HVR-H3), according to U.S.Pat. No. 8,535,675:

HVR-L1 RSSETLVHSSGNTYLE (SEQ ID NO: 75) HVR-L2 RVSNRFS (SEQ ID NO: 76)HVR-L3 FQGSFNPLT (SEQ ID NO: 77) HVR-H1 GFSFSDFAMS (SEQ ID NO: 78)HVR-H2 ATIGRVAFHTYYPDSMKG (SEQ ID NO: 79) HVR-H3 ARHRGFDVGHFDF(SEQ ID NO: 80)

The anti-NaPi2b antibodies of ADC in Table 5 comprise the light chainvariable region (VL) shown in SEQ ID NO: 81 and the heavy chain variableregion (VH) shown in SEQ ID NO: 82.

(SEQ ID NO: 81) DIQMTQSPSSLSASVGDRVTITCRSSETLVHSSGNTYLEWYQQKPGKAPKLLIYRVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSFNP LTFGQGTKVEIKR(SEQ ID NO: 82) EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFAMSWVRQAPGKGLEWVATIGRVAFHTYYPDSMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHR GFDVGHFDFWGQGTLVTVSS

In some embodiments, the invention provides an anti-NaPi2b antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:79; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:80; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:75; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:76; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:77.

In one aspect, the invention provides an anti-NaPi2b antibody comprisingat least one, at least two, or all three VH HVR sequences selected from(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:79; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:80. In oneembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:80. In another embodiment, the antibody comprisesHVR-H3 comprising the amino acid sequence of SEQ ID NO:80 and HVR-L3comprising the amino acid sequence of SEQ ID NO:77. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:80, HVR-L3 comprising the amino acid sequence ofSEQ ID NO:77, and HVR-H2 comprising the amino acid sequence of SEQ IDNO:79. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:78; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:79; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:80.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:76; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:77. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:75; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:76; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:77.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:78, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:79, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:80; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:75, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:76, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:77.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:78; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:79; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:80; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:75; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:76; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:77.

In any of the above embodiments, an anti-NaPi2b antibody is humanized.In one embodiment, an anti-NaPi2b antibody comprises HVRs as in any ofthe above embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-NaPi2b antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:82. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO:82 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-NaPi2b antibody comprising that sequenceretains the ability to bind to NaPi2b. In certain embodiments, a totalof 1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO:82. In certain embodiments, a total of 1 to 5 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:82. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-NaPi2bantibody comprises the VH sequence of SEQ ID NO:82, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:78, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:79, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:80.

In another aspect, an anti-NaPi2b antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:81. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ IDNO:81 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-NaPi2b antibody comprising that sequence retains the ability tobind to NaPi2b. In certain embodiments, a total of 1 to 10 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO:81. Incertain embodiments, a total of 1 to 5 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:81. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-NaPi2bantibody comprises the VL sequence of SEQ ID NO:81 includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:76; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:77.

In another aspect, an anti-NaPi2b antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:82 and SEQ ID NO:81, respectively, including post-translationalmodifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-NaPi2b antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-NaPi2b antibody comprising a VH sequence of SEQ IDNO:82 and a VL sequence of SEQ ID NO:81.

In a further aspect of the invention, an anti-NaPi2b antibody accordingto any of the above embodiments is a monoclonal antibody, including ahuman antibody. In one embodiment, an anti-NaPi2b antibody is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, the antibody is a substantially fulllength antibody, e.g., an IgG1 antibody, IgG2a antibody or otherantibody class or isotype as defined herein.

In a further aspect, an anti-NaPi2b antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described below.

Anti-CD22 Antibodies

The anti-CD22 antibodies of ADC in Table 5 comprise three light chainhypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavy chainhypervariable regions (HVR-H1, HVR-H2 and HVR-H3), according to U.S.Pat. No. 8,226,945:

HVR-L1 RSSQSIVHSVGNTFLE (SEQ ID NO: 1) HVR-L2 KVSNRFS (SEQ ID NO: 2)HVR-L3 FQGSQFPYT (SEQ ID NO: 3) HVR-H1 GYEFSRSWMN (SEQ ID NO: 4) HVR-H2GRIYPGDGDTNYSGKFKG (SEQ ID NO: 5) HVR-H3 DGSSWDWYFDV (SEQ ID NO: 6)Anti-MUC16 Antibodies

The anti-MUC16 antibodies of ADC in Table 5 comprise three light chainhypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavy chainhypervariable regions (HVR-H1, HVR-H2 and HVR-H3), according to U.S.Pat. No. 7,989,595:

HVR-L1 KASDLIHNWLA (SEQ ID NO: 7) HVR-L2 YGATSLET (SEQ ID NO: 8) HVR-L3QQYWTTPFT (SEQ ID NO: 9) HVR-H1 GYSITNDYAWN (SEQ ID NO: 10) HVR-H2GYISYSGYTTYNPSLKS (SEQ ID NO: 11) HVR-H3 ARWTSGLDY (SEQ ID NO: 12)

The anti-MUC16 antibodies of ADC in Table 5 comprise the variable heavychain sequence of SEQ ID NO: 13 and the variable light chain sequence ofSEQ ID NO: 14.

(SEQ ID NO: 13) E V Q L V E S G G G L V Q P G G S L R L S C A A SG Y S I T N D Y A W N W V R Q A P G K G L E W V GY I S Y S G Y T T Y N P S L K S R F T I S R D T SK N T L Y L Q M N S L R A E D T A V Y Y C A R W TS G L D Y W G Q G T L V T V S S (SEQ ID NO: 14)D I Q M T Q S P S S L S A S V G D R V T I T C K AS D L I H N W L A W Y Q Q K P G K A P K L L I Y GA T S L E T G V P S R F S G S G S G T D F T L T IS S L Q P E D F A T Y Y C Q Q Y W T T P F T F G Q G T K V E I K R

In one embodiment, the anti-MUC16 antibody of an ADC of the invention isa cysteine-engineered, ThioMab comprising one or more free cysteineamino acid residues located in a light chain sequence selected from SEQID NOs: 15-32, or in a heavy chain sequence selected from SEQ ID NOs:33-46:

EVQLCESGGG SEQ ID NO: 15 LRLSCCASGYS SEQ ID NO: 16 MNSLRCEDTAVSEQ ID NO: 17 TLVTVCSASTK SEQ ID NO: 18 VTVSSCSTKGP SEQ ID NO: 19VSAASCKGPSV SEQ ID NO: 20 WYVDGCEVHNA SEQ ID NO: 21 KGFVPCDIAVESEQ ID NO: 22 PPVLDCGDSFF SEQ ID NO: 23 DVQLCESGPG SEQ ID NO: 24LSLTCCVTGYS SEQ ID NO: 25 LNSVTCEDTAT SEQ ID NO: 26 TLVTVCSASTKSEQ ID NO: 27 VTVSSCSTKGP SEQ ID NO: 28 VSAASCKGPSV SEQ ID NO: 29WYVDGCEVHNA SEQ ID NO: 30 KGFVPCDIAVE SEQ ID NO: 31 PPVLDCDGSFFSEQ ID NO: 32 SLSASCGDRVT SEQ ID NO: 33 EIKRTCAAPSV SEQ ID NO: 34TVAAPCVFIFP SEQ ID NO: 35 FIFPPCDEQLK SEQ ID NO: 36 DEQLKCGTASVSEQ ID NO: 37 VTEQDCKDSTY SEQ ID NO: 38 GLSSPCTKSFN SEQ ID NO: 39FLSVSCGGRVT SEQ ID NO: 40 EIKRTCAAPSV SEQ ID NO: 41 TVAAPCVFIFPSEQ ID NO: 42 FIFPPCDEQLK SEQ ID NO: 43 DEQLKCGTASV SEQ ID NO: 44VTEQDCKDSTY SEQ ID NO: 45 GLSSPCTKSFN SEQ ID NO: 46

In one embodiment, the anti-MUC16 cysteine-engineered, ThioMab is ahumanized antibody comprising the heavy chain sequence of SEQ ID NO:47.

(SEQ ID NO: 47) EVQLVESGGGLVQPGGSLRLSCAASGYSITNDYAWNWVRQAPGKGLEWVGYISYSGYTTYNPSLKSRFTISRDISKNTLYLQMNSLRAEDTAVYYCARWTSGLDYWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In one embodiment, the anti-MUC16 cysteine-engineered, ThioMab is ahumanized antibody comprising the light chain sequence of SEQ ID NO:48.

(SEQ ID NO: 48) DIQMTQSPSSLSASVGDRVTITCKASDLIHNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWTTPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

In one embodiment, the anti-MUC16 cysteine-engineered, ThioMab is achimeric antibody comprising the heavy chain sequence of SEQ ID NO:49.

(SEQ ID NO: 49) DVQLQESGPGLVNPSQSLSLICTVTGYSITNDYAWNWIRQFPGNKLEWMGYINYSGYTTYNPSLKSRISITRDTSKNQFFLHLNSVTTEDTATYYCARWDGGLTYWGQGTLVTVSACSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In one embodiment, the anti-MUC16 cysteine-engineered, ThioMab is achimeric antibody comprising the light chain sequence of SEQ ID NO:50.

(SEQ ID NO: 50) DIQMTQSSSFLSVSLGGRVTITCKASDLIHNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGNDYTLSIASLQTEDAATYYCQQYWTTPFTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECAnti-HER2 Antibodies

In certain embodiments, ADC of Table 5 comprise anti-HER2 antibodies. Inone embodiment of the invention, an anti-HER2 antibody of an ADC of theinvention comprises a humanized anti-HER2 antibody, e.g., huMAb4D5-1,huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7and huMAb4D5-8, as described in Table 3 of U.S. Pat. No. 5,821,337.Those antibodies contain human framework regions with thecomplementarity-determining regions of a murine antibody (4D5) thatbinds to HER2. The humanized antibody huMAb4D5-8 is also referred to astrastuzumab, commercially available under the tradename HERCEPTIN®. Inanother embodiment of the invention, an anti-HER2 antibody of an ADC ofthe invention comprises a humanized anti-HER2 antibody, e.g., humanized2C4, as described in U.S. Pat. No. 7,862,817. An exemplary humanized 2C4antibody is pertuzumab, commercially available under the tradenamePERJETA®.

The cysteine-engineered Thiomab antibodies used to prepare the ADC ofTable 5 have a cysteine residue introduced at the 118-alanine site (EUnumbering) of the heavy chain. This site is numbered 121 by Sequentialnumbering or 114 by Kabat numbering.

Anti-CD33 Antibodies

The anti-CD33 antibody 15G15.33 of ADC in Table 5 comprises three lightchain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavychain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3)

HVR-L1 RSSQSLLHSNGYNYLD (SEQ ID NO: 51) HVR-L2 LGVNSVS (SEQ ID NO: 52)HVR-L3 MQALQTPWT (SEQ ID NO: 53) HVR-H1 NHAIS (SEQ ID NO: 54) HVR-H2GIIPIFGTANYAQKFQG (SEQ ID NO: 55) HVR-H3 EWADVFD (SEQ ID NO: 56)

The anti-CD33 antibody 15G15.33 of ADC in Table 5 comprises the lightchain variable region of SEQ ID NO:57 and/or the heavy chain variableregion of SEQ ID NO:58.

(SEQ ID NO: 57) EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGVNSVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP WTFGQGTKVEIK(SEQ ID NO: 58) QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAFMELSSLRSEDTAVYYCAREW ADVFDIWGQGTMVTVSSAnti-CD33 Antibody 9C3 and Other Embodiments

9C3-HVR L1 RASQGIRNDLG (SEQ ID NO: 59) 9C3-HVR L2 AASSLQS(SEQ ID NO: 60) 9C3-HVR L3 LQHNSYPWT (SEQ ID NO: 61) 9C3-HVR H1 GNYMS(SEQ ID NO: 62) 9C3-HVR H2 LIYSGDSTYYADSVKG (SEQ ID NO: 63) 9C3-HVR H3DGYYVSDMVV (SEQ ID NO: 64) 9C3 V_(L) (SEQ ID NO: 65)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQ GTKLEIK 9C3 V_(H)(SEQ ID NO: 66) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFNISRDISKNTVYLQMNSLRVEDTAVYYCVRDGY YVSDMVVWGKGTTVTVSS9C3.2 V_(L) (SEQ ID NO: 67)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQ GTKLEIK 9C3.2 V_(H)(SEQ ID NO: 68) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFTISRDISKNTVYLQMNSLRVEDTAVYYCVRDGY YVSDMVVWGKGTTVTVSS9C3.3 V_(L) (SEQ ID NO: 69)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQ GTKLEIK 9C3.3 V_(H)(SEQ ID NO: 70) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFSISRDISKNTVYLQMNSLRVEDTAVYYCVRDGY YVSDMVVWGKGTTVTVSS9C3.4 V_(L) (SEQ ID NO: 71)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQ GTKLEIK 9C3.4 V_(H)(SEQ ID NO: 72) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFAISRDISKNTVYLQMNSLRVEDTAVYYCVRDGY YVSDMVVWGKGTTVTVSS

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:62; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:59; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:60; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:61.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:62; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:63; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:64. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:64 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:61. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:64, HVR-L3 comprising the amino acid sequence of SEQ ID NO:61, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:63. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:62; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:63; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:64.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:59; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:60; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:61. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:59; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:60; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:61.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:62, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:63, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:64; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:59, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:60, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:61.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:62; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:63; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:59; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:60; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:61.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ IDNO:72. In certain embodiments, a VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acidsequence of SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ IDNO:72 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70, and/or SEQ ID NO:72. In certain embodiments, a total of 1to 5 amino acids have been substituted, inserted and/or deleted in SEQID NO:66, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:72. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-CD33 antibodycomprises the VH sequence of SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,and/or SEQ ID NO:72, including post-translational modifications of thatsequence. In a particular embodiment, the VH comprises one, two or threeHVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQID NO:62, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:63,and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:64.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:65, SEQ ID NO:67, SEQID NO:69, and/or SEQ ID NO:71. In certain embodiments, a VL sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO:65, SEQ ID NO:67, SEQID NO:69, and/or SEQ ID NO:71 contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-CD33 antibody comprising that sequence retains theability to bind to CD33. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, and/or SEQ ID NO:71. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,and/or SEQ ID NO:71. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-CD33 antibody comprises the VL sequence ofSEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and/or SEQ ID NO:71, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:59; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:60; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:61.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:66 and SEQ ID NO:65, respectively, including post-translationalmodifications of those sequences. In one embodiment, the antibodycomprises the VH and VL sequences in SEQ ID NO:68 and SEQ ID NO:67,respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:70 and SEQ ID NO:69, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:72 and SEQID NO:71, respectively, including post-translational modifications ofthose sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:72 and a VL sequenceof SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and/or SEQ ID NO:71,respectively.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells having receptorproteins, e.g. HER2, to the antibody of the ADC in a cell culturemedium; culturing the cells for a period from about 6 hours to about 5days; and measuring cell viability. Cell-based in vitro assays were usedto measure viability (proliferation), cytotoxicity, and induction ofapoptosis (caspase activation) of the ADC of the invention.

The in vitro potency of antibody-drug conjugates (ADC) was measured by acell proliferation assay (Example 21). The ADC showed surprising andunexpected potency in inhibition of tumor cell proliferation. Potency ofthe ADC was correlated with target antigen expression of the cells. Thedata of FIGS. 25-30 demonstrate the tested conjugates are capable ofbinding to the specific antigen expressed on the surface of cells andcausing the death of those cells in vitro.

The CellTiter-Glo® Luminescent Cell Viability Assay is a commerciallyavailable (Promega Corp., Madison, Wis.), homogeneous assay method basedon the recombinant expression of Coleoptera luciferase (U.S. Pat. Nos.5,583,024; 5,674,713; 5,700,670). This cell proliferation assaydetermines the number of viable cells in culture based on quantitationof the ATP present, an indicator of metabolically active cells (Crouchet al (1993) J. Immunol. Meth. 160:81-88; U.S. Pat. No. 6,602,677). TheCellTiter-Glo® Assay was conducted in 96 well format, making it amenableto automated high-throughput screening (HTS) (Cree et al (1995)AntiCancer Drugs 6:398-404). The homogeneous assay procedure involvesadding the single reagent (CellTiter-Glo® Reagent) directly to cellscultured in serum-supplemented medium. Cell washing, removal of mediumand multiple pipetting steps are not required. The system detects as fewas 15 cells/well in a 384-well format in 10 minutes after adding reagentand mixing. The cells may be treated continuously with ADC, or they maybe treated and separated from ADC. Generally, cells treated briefly,i.e. 3 hours, showed the same potency effects as continuously treatedcells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons.

Cell-based in vitro assays are used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the ADC of the invention. Generally, the cytotoxic orcytostatic activity of an antibody-drug conjugate (ADC) is measured by:exposing mammalian cells expressing antigen such as Her2 or MUC16polypeptide to ADC in a cell culture medium; culturing the cells for aperiod from about 6 hours to about 5 days; and measuring cell viability.Mammalian cells useful for cell proliferation assays for anti-MUC16 ADCinclude: (1) a MUC16 polypeptide-expressing cell line OVCAR-3; (2) aPC3-derived cell line engineered to stably express a portion of theMUC16 polypeptide on its cell surface (PC3/MUC16); (3) the parental PC3cell line that does not express the MUC16 polypeptide; and (4) a PC3cell line that does not express MUC16 polypeptide but carries the vectorused to drive exogenous MUC16 expression (PC3/neo).

FIG. 25 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 3 days versus concentrations (μg/ml)of Thio hu anti-CD22 HC A121C-MC-vc-PAB-(CBI dimer) 101 and Thio huanti-Her2 HC A121C-MC-vc-PAB-(CBI dimer) 102. The Her2 antigen is highlyexpressed in SK-BR-3 cells. The anti-Her2 ADC 102 shows linear, non-doseresponse cell-killing activity whereas control, off-target anti-CD22 ADC101 shows less activity.

FIG. 26 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 3 days versus concentrations (μg/ml)of Thio Hu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI-PBD) 103 and Thio HuAnti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI-PBD) 104. The anti-Her2 ADC 103shows linear, non-dose response cell-killing activity whereas control,off-target anti-CD22 ADC 104 shows less activity.

FIG. 27 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 3 days versus concentrations (μg/ml)of Thio Hu Anti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI dimer) 116 and ThioHu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI dimer) 117. The anti-Her2 ADC117 shows linear, non-dose response cell-killing activity whereascontrol, off-target anti-CD22 ADC 116 shows less activity.

FIG. 28 shows the efficacy of antibody-drug conjugates in a plot ofEOL-1 in vitro cell viability at 3 days versus concentrations (μg/ml) ofThio Hu Anti-CD33 15G15.33 HC A118C-MC-MMED-(CBI dimer phos) 125 whichshows modest dose response cell-killing activity.

FIG. 29 shows the efficacy of antibody-drug conjugates in a plot ofEOL-1 in vitro cell viability at 3 days versus concentrations (μg/ml) ofThio Hu Anti-MUC16 3A5 HC A118C-MC-ED-(CBI dimer DVB diphos) 126 andThio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBI dimer DVB diphos) 127.The anti-CD33 ADC 127 shows potent, dose responsive cell-killingactivity whereas control, off-target anti-MUC16 ADC 126 shows lessactivity.

FIG. 30 shows the efficacy of antibody-drug conjugates in a plot ofEOL-1 in vitro cell viability at 3 days versus concentrations (μg/ml) ofThio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBI dimer DVB diphos) 127,Thio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBI dimer DVB phos) 129, andThio Hu Anti-MUC16 3A5 HC A118C-MC-ED-(CBI dimer DVB phos) 130. Theanti-CD33 ADC 127 and 129 shows potent, dose responsive cell-killingactivity whereas control, off-target anti-MUC16 ADC 130 shows lessactivity.

In Vivo Efficacy

The in vivo efficacy of antibody-drug conjugates (ADC) of the inventioncan be measured by tumor xenograft studies in mice (Example 22). The invivo efficacy of antibody-drug conjugates (ADC) was measured tumorgrowth inhibition in mice (Example 21). The ADC showed surprising andunexpected potency in inhibition of tumor growth. Efficacy of the ADCwas correlated with target antigen expression of the tumor cells.

The efficacy of antibody-drug conjugates were measured in vivo byimplanting allografts or xenografts of cancer cells in rodents andtreating the tumors with ADC. Variable results are to be expecteddepending on the cell line, the specificity of antibody binding of theADC to receptors present on the cancer cells, dosing regimen, and otherfactors. The in vivo efficacy of the ADC was measured using a transgenicexplant mouse model expressing moderate to high levels of atumor-associated antigen, such as Her2, MUC16, and CD33. Subjects weretreated once with ADC and monitored over 3-6 weeks to measure the timeto tumor doubling, log cell kill, and tumor shrinkage. Follow updose-response and multi-dose experiments were conducted.

For example, the in vivo efficacy of an anti-HER2 ADC of the inventioncan be measured by a high expressing HER2 transgenic explant mouse model(Phillips et al (2008) Cancer Res. 68:9280-90). An allograft ispropagated from the Fo5 mmtv transgenic mouse which does not respond to,or responds poorly to, HERCEPTIN® therapy. Subjects were treated oncewith ADC at certain dose levels (mg/kg) and placebo buffer control(Vehicle) and monitored over two weeks or more to measure the time totumor doubling, log cell kill, and tumor shrinkage.

FIG. 31 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: (1) Vehicle: Histidine Buffer #8: 20 mMHistidine Acetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (2) Thio HuAnti-CD22 10F4v3 HC A118C-MC-MMED-(CBI dimer phos) 110, (3) Thio HuAnti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI dimer MePip) 108, (4) Thio HuAnti-CD22 10F4v3 HC A118C-MC-vc-PAB-(CBI dimer phos) 111, (5) Thio HuAnti-Her2 4D5 HC A118C-MC-MMED-(CBI dimer phos) 109, (6) Thio HuAnti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI dimer MePip) 107, (7) Thio HuAnti-Her2 4D5 HC A118C-MC-vc-PAB-(CBI dimer phos) 112. ADC were dosed at10 mg/kg.

FIG. 32 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: (1) Vehicle: Histidine Buffer #8: 20 mMHistidine Acetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (2) Thio HuAnti-CD22 10F4v3 HC A118C-DSE-(CBI dimer phos) 120, 10 mg/kg, (3) ThioHu Anti-CD22 10F4v3 HC A118C-DSE-(CBI dimer phos) 122, 10 mg/kg, (4)Thio Hu Anti-CD22 10F4v3 HC All 18C-MC-vc-PAB-(N10,PBD-CBI MePip) 124,10 mg/kg, (5) Thio Hu Anti-Her2 4D5 HC A118C-DSE-(CBI dimer phos) 119, 3mg/kg, (6) Thio Hu Anti-Her2 4D5 HC A118C-DSE-(CBI dimer phos) 119, 10mg/kg, (7) Thio Hu Anti-Her2 4D5 HC A118C-DSP-(CBI dimer phos) 121, 3mg/kg, (8) Thio Hu Anti-Her2 4D5 HC A118C-DSP-(CBI dimer phos) 121, 10mg/kg (9) Thio Hu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(N10,PBD-CBI MePip)123, 3 mg/kg, (10) Thio Hu Anti-Her2 4D5 HC A118C-MC-vc-PAB-(N10,PBD-CBIMePip) 123, 10 mg/kg.

FIG. 33 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in OVCAR3X2.1 human ovariantumors inoculated into C.B-17 SCID mice after dosing once IV with: (1)Vehicle: Histidine Buffer #8: 20 mM Histidine Acetate, pH 5.5, 240 mMSucrose, 0.02% PS 20, (2) Thio Hu Anti-CD33 15G15.33 HC A118C-MC-ED-(CBIdimer DVB diphos) 127, 3 mg/kg, (3) Thio Hu Anti-MUC16 3A5 HCA118C-MC-ED-(CBI dimer DVB diphos) 126, 3 mg/kg, (4) Thio Hu Anti-MUC163A5 HC A118C-MC-ED-(CBI dimer DVB diphos) 126, 1 mg/kg.

The anti-MUC16 antibody of ADC 126, and including 3A5 and 11D10variants, have been described in WO 2007/001851; U.S. Pat. No.7,989,595; 8,449,883, the contents of which are incorporated byreference. The 3A5 monoclonal antibody binds multiple sites of the MUC16polypeptide with 433 pM affinity by OVCAR-3 Scatchard analysis (Chen etal (2007) Cancer Res. 67(10): 4924-4932)

FIG. 34 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in HL-60 human acutemyeloid leukemia inoculated into C.B-17 SCID mice after dosing once IVat 20 μg/m2 with: (1) Vehicle: Histidine Buffer #8: 20 mM HistidineAcetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (2) Thio Hu Anti-CD3315G15.33 HC A118C-MC-MMED-(CBI dimer phos) 125, (3) Thio Hu Anti-CD3315G15.33 HC A118C-MC-ED-(CBI dimer DVB diphos) 127, (4) Thio HuAnti-MUC16 3A5 HC A118C-MC-MMED-(CBI dimer phos) 128, (5) Thio HuAnti-MUC16 3A5 HC A118C-MC-ED-(CBI dimer DVB diphos) 126.

Administration of Antibody-Drug Conjugates

The antibody-drug conjugates (ADC) of the invention may be administeredby any route appropriate to the condition to be treated. The ADC willtypically be administered parenterally, i.e. infusion, subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural.

Pharmaceutical Formulations

Pharmaceutical formulations of therapeutic antibody-drug conjugates(ADC) of the invention are typically prepared for parenteraladministration, i.e. bolus, intravenous, intratumor injection with apharmaceutically acceptable parenteral vehicle and in a unit dosageinjectable form. An antibody-drug conjugate (ADC) having the desireddegree of purity is optionally mixed with pharmaceutically acceptablediluents, carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the formof a lyophilized formulation or an aqueous solution.

Antibody-Drug Conjugate Treatments

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignantsolid tumors and hematological disorders such as leukemia and lymphoidmalignancies. Others include neuronal, glial, astrocytal, hypothalamic,glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory,angiogenic and immunologic, including autoimmune, disorders.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatmentinclude rheumatologic disorders (such as, for example, rheumatoidarthritis, Sjögren's syndrome, scleroderma, lupus such as systemic lupuserythematosus (SLE) and lupus nephritis, polymyositis/dermatomyositis,cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriaticarthritis), osteoarthritis, autoimmune gastrointestinal and liverdisorders (such as, for example, inflammatory bowel diseases (e.g.,ulcerative colitis and Crohn's disease), autoimmune gastritis andpernicious anemia, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, and celiac disease), vasculitis (suchas, for example, ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and polyarteriitis), autoimmuneneurological disorders (such as, for example, multiple sclerosis,opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica,Parkinson's disease, Alzheimer's disease, and autoimmunepolyneuropathies), renal disorders (such as, for example,glomerulonephritis, Goodpasture's syndrome, and Berger's disease),autoimmune dermatologic disorders (such as, for example, psoriasis,urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneouslupus erythematosus), hematologic disorders (such as, for example,thrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, autoimmune hearing diseases (such as, forexample, inner ear disease and hearing loss), Behcet's disease,Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders(such as, for example, diabetic-related autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), Addison's disease, andautoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the disordersdescribed above is provided. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, blister pack, etc. The containers may be formed from a varietyof materials such as glass or plastic. The container holds anantibody-drug conjugate (ADC) composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an ADC. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

EXAMPLES Example 1(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(2-bromo-N-methylacetamido)ethyl(methyl)carbamate 51

Following the procedure of L. F. Tietze, J. M. von Hof, M. Müller, B.Krewer, I. Schuberth, Angew. Chem. Int. Ed. (2010) 49:7336-7339,(S)-tert-Butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a wasdeprotected with HCl and acylated with glutaroyl dichloride (FIG. 1).Instead of preparative HPLC, reaction solvent was removed under vacuumand the resultant residue was triturated with methanol to give1,5-bis((S)-1-(Chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione51b as an off-white solid (yield 51%). R_(f)=0.50 (ethylacetate/petroleum ether=2:1). NMR and MS data are identical to thereported value. [α]_(D) ²⁶=−46.3° (c=0.41, DMA).

To a solution of 51b (650 mg, 1.15 mmol) in DMA (5 mL) cooled in an icebath was added DIPEA (0.40 mL, 2.31 mmol) and 4-nitrophenylchloroformate (302 mg, 1.50 mmol). After the mixture was allowed to warmup to room temperature and stirred overnight, tert-butylmethyl(2-(methylamino)ethyl)carbamate (652 mg, 3.00 mmol) was added. Themixture was stirred at room temperature for 7 h and then redistributedbetween ethyl acetate and cold dilute aqueous NaHCO₃. The aqueous phasewas extracted with ethyl acetate three times. The combined organicextracts were washed with water followed by brine, dried over anhydrousNa₂SO₄, and filtered through celite. The solvent was removed and theresidue was further purified by column chromatography using gradientmixtures of ethyl acetate and petroleum ether (v/v 1:1 to 4:1) and thenethyl acetate only as eluents to give Boc(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylmethyl(2-(methylamino)ethyl)carbamate 51c as an off-white solid (359 mg,40%); mp 157° C. (dec.). [α]_(D) ²⁶=−45.4° (c=1.08, ethyl acetate). ¹HNMR (DMSO) (mixture of rotamers) δ 10.35 (s, 1H), 8.25 (s, 1H), 8.08 (d,J=8.2 Hz, 1H), 8.02 (s, 1H), 7.87-7.77 (m, 2H), 7.58 (t, J=7.6 Hz, 1H),7.50-7.42 (m, 2H), 7.31 (t, J=7.9 Hz, 1H), 4.42 (t, J=9.7 Hz, 1H),4.36-4.31 (m, 2H), 4.25 (d, J=10.3 Hz, 1H), 4.19-4.13 (m, 2H), 4.05 (dd,J=2.9, 11.0 Hz, 1H), 3.98 (dd, J=2.6, 10.9 Hz, 1H), 3.92 (dd, J=7.2,10.9 Hz, 1H), 3.79 (dd, J=8.5, 10.2 Hz, 1H), 3.69 (br s, 1H), 3.52-3.42(m, 3H), 3.21-2.79 (m, 6H, 2NMe), 2.75-2.67 (m, 2H), 2.64-2.58 (m, 2H),2.00-1.93 (m, 2H), 1.44-1.35 (m, 9H, Bu^(t)) ppm. HRMS (ESI) found m/z777.2801 (M+H). C₄₁H₄₇Cl₂N₄O₇ requires 777.2816.

Also obtained from the same chromatographic separation were bis-Bocprotected 51d and unprotected recovered starting material 51b as a whitesolid (15 mg, 2%). 51d (429 mg, 37%) was obtained as an off-white solid.[α]_(D) ²⁶=−32.0° (c=1.00, ethyl acetate). ¹H NMR (DMSO) (mixture ofrotamers) δ 8.25 (s, 2H), 7.95 (d, J=8.4 Hz, 2H), 7.87-7.76 (m, 2H),7.58 (t, J=7.6 Hz, 2H), 7.44 (t, J=7.6 Hz, 2H), 4.41 (t, J=9.8 Hz, 2H),4.32 (br s, 2H), 4.24 (d, J=10.6 Hz, 2H), 4.06-4.03 (m, 2H), 3.98 (dd,J=7.3, 10.8 Hz, 2H), 3.69 (br s, 2H), 3.52-3.43 (m, 6H), 3.21-2.79 (m,12H, 4NMe), 2.75-2.69 (m, 2H), 2.66-2.58 (m, 2H), 1.99-1.95 (m, 2H),1.44-1.35 (m, 18H, 2Bu^(t)) ppm. HRMS (ESI) found m/z 1013.3991 (M+Na).C₅₁H₆₄Cl₂N₆NaO₁₀ requires 1013.3991.

To a solution of 51c (200 mg, 0.26 mmol) in DCM (3 mL) cooled in an icebath was added trifluoroacetic acid, TFA (1.5 mL) dropwise. The mixturewas allowed to warm up to room temperature and stirred for 2 h. Allvolatile components were removed to give crude trifluoroacetate salt of(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylmethyl(2-(methylamino)ethyl)carbamate 51e as an off-white solid, whichwas used directly. ¹H NMR (DMSO) (mixture of rotamers) δ 10.36 (s, 1H),8.64 (br s, 1H), 8.48 (br s, 1H), 8.35 (s, 1H), 8.08 (d, J=8.1 Hz, 1H),8.01 (s, 1H), 7.97 (d, J=8.4 Hz, 1H), 7.94-7.88 (m, 1H), 7.78 (d, J=8.4Hz, 1H), 7.61-7.57 (m, 2H), 7.51-7.45 (m, 2H), 7.34-7.30 (m, 1H), 4.43(t, J=9.8 Hz, 1H), 4.36-4.31 (m, 2H), 4.26 (d, J=10.4 Hz, 1H), 4.18-4.14(m, 2H), 4.06 (dd, J=2.9, 11.0 Hz, 1H), 4.00 (dd, J=2.7, 10.8 Hz, 1H),3.94 (dd, J=7.5, 11.0 Hz, 1H), 3.89-3.84 (m, 1H), 3.79 (dd, J=8.1, 10.8Hz, 1H), 3.63 (t, J=5.7 Hz, 1H), 3.33-3.30 (m, 6H, 2NMe), 2.78-2.55 (m,6H), 2.00-1.94 (m, 2H) ppm. HRMS (ESI) found m/z 677.2306 (M+H).C₃₆H₃₉Cl₂N₄O₅ requires 677.2292.

At −5° C., to a solution of 51e in THF (4 mL), was added a drop of DIPEAfollowed by bromoacetyl bromide (34 μL, 0.39 mmol) slowly and then therest of DIPEA (448 μL, 2.57 mmol in total). The mixture was allowed towarm up to room temperature and stirred for 1 h. All volatile componentswere removed by rotary evaporator and then high vacuum pump. Theresultant residue was stirred with ethyl acetate and the insoluble solidwas filtered off before the filtrate was loaded on a chromatographycolumn. Gradient mixtures of ethyl acetate and petroleum ether (v/v 1:4to 1:1) were used as eluents to give(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(2-bromo-N-methylacetamido)ethyl(methyl)carbamate 51 as an off-whitesolid (80 mg, 39%); mp 167-170° C. [α]_(D) ²⁶=−64.0° (c=0.25, ethylacetate). ¹H NMR (DMSO) (mixture of rotamers) δ 10.39 (s, 1H), 8.25-8.20(m, 1H), 8.08 (d, J=8.3 Hz, 1H), 8.01 (s, 1H), 7.96 (d, J=8.3 Hz, 1H),7.89 (d, J=7.4 Hz, 0.34H), 7.82 (d, J=7.4 Hz, 0.66H), 7.78 (d, J=8.4 Hz,1H), 7.58 (t, J=7.6 Hz, 1H), 7.51-7.45 (m, 2H), 7.31 (t, J=7.6 Hz, 1H),4.42 (t, J=9.6 Hz, 1H), 4.36-4.31 (m, 1H), 4.24 (d, J=10.7 Hz, 1H),4.18-4.09 (m, 3H including CH₂Br), 4.07-4.03 (dd, J=2.8, 10.9 Hz, 1H),4.00-3.97 (dd, J=2.6, 10.8 Hz, 1H), 3.96-3.91 (m, 1H), 3.82-3.75 (m,1H), 3.70-3.68 (m, 1H), 3.58-3.43 (m, 3H), 3.26-2.89 (m, 6H, 2NMe),2.78-2.67 (m, 2H), 2.65-2.55 (m, 2H), 1.99-1.92 (m, 2H) ppm. HRMS (ESI)found m/z 819.1316 (M+Na). C₃₈H₃₈BrCl₂N₄O₆ requires 819.1322.

Example 2 (11S,11aS)-tert-butyl8-(6-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate52

Following the experimental procedures of Example 6, linker-drugintermediate 52 was prepared (FIGS. 7 and 8). HPLC: 96.7% pure; mp 210°C. (dec.); ¹H NMR [(CD₃)₂SO] δ 10.03 (s, exchangeable with D₂O, 1H),8.23-8.11 (m, 2H), 8.09 (d, J=7.3 Hz, exchangeable with D₂O, 1H), 7.85(d, J=8.5 Hz, 1H), 7.80 (d, J=8.3 Hz, exchangeable with D₂O, 1H), 7.66(d, J=8.6 Hz, 2H), 7.59-7.44 (m, 3H), 7.38 (br t, J=7.6 Hz, 1H), 7.04(s, 1H), 6.99 (s, 2H), 6.69 (s, 1H), 6.38 (br s, exchangeable with D₂O,1H), 5.99 (t, J=5.5 Hz, exchangeable with D₂O, 1H), 5.49-5.34 (m, 3H,reduced to 1H as d after D₂O, J=9.5 Hz), 5.20 (s, 2H), 4.44-4.30 (m,2H), 4.26-4.13 (m, 3H), 4.10-3.91 (m, 3H), 3.88-3.76 (m, 1H), 3.79 (s,3H), 3.53-3.44 (m, 1H), 3.41-3.20 (m, partially obscured by water peak,4H), 3.09-2.88 (m, 2H), 2.66-2.42 (m, partially obscured by DMSO peak,2H), 2.25-1.24 (m, 21H), 1.31 (s, 9H), 1.24-1.11 (m, 2H), 0.86 (d, J=6.8Hz, 3H), 0.82 (d, J=6.7 Hz, 3H). Anal. (C₅₅H₇₁ClN₈O₁₁.H₂O) Calc: C,61.53; H, 6.85; N, 10.44. Found: C, 61.39; H, 7.11; N, 10.15.

Example 3N—((R)-1-(chloromethyl)-3-(5-((R)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide53

Acetic acid (50 mL) was added to a stirred solution of (R)-tert-butyl1-(chloromethyl)-5-(diphenylmethyleneamino)-1H-benzo[e]indole-3(2H)-carboxylate53a (1.00 g, 2.01 mmol) in THF-H₂O (150 mL/75 mL) at room temperature(r.t.) and the mixture stirred overnight (FIG. 2). After 19.5 h the THFwas removed under vacuum, the mixture diluted with EtOAc, the layerswell shaken and then separated. The organic layer was washed withsaturated aqueous NaHCO₃ (4×), dried (Na₂SO₄) and solvent removed undervacuum. Purification by column chromatography on silica gel usinghexanes:EtOAc 100:0 to 90:10 gave (R)-tert-butyl5-amino-1-(chloromethyl)-1H-benzo[e]indole-3 (2H)-carboxylate 53b (382mg, 57%) as an orange gel-like solid. ¹H NMR δ (400 MHz, DMSO-d₆) 8.01(d, J=8.4 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.42-7.38 (m, 1H), 7.37 (brs, 1H), 7.22-7.18 (m, 1H), 5.91 (s, 2H), 4.08-3.91 (m, 4H), 3.66 (dd,J=10.6, 8.2 Hz, 1H), 1.53 (s, 9H).

A mixture of 53b (380 mg, 1.14 mmol),6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (361 mg, 1.71mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDCI.HCl, 765 mg, 3.99 mmol) and para-toluenesulfonic acid (TsOH, 49mg, 0.285 mmol) in dry DMA (10 mL) was stirred at r.t. overnight, undernitrogen. After 17 h the solvent was removed under vacuum. The crudeproduct was purified by column chromatography on silica gel usingDCM:hexanes 75:25 to 100:0, then DCM:MeOH 99:1 to 97:3 and theproduct-containing fractions evaporated to dryness. The resultingmaterial was then dissolved in EtOAc and the organic layer washed withH₂O (2×), dried (Na₂SO₄) and solvent removed to give (R)-tert-butyl1-(chloromethyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-1H-benzo[e]indole-3(2H)-carboxylate53c (282 mg, 47%) as a yellow solid. ¹H NMR δ (400 MHz, DMSO-d₆) 9.86(s, 1H), 8.24 (br s, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.88 (d, J=8.3 Hz,1H), 7.55-7.51 (m, 1H), 7.43-7.39 (m, 1H), 7.01 (s, 2H), 4.21-4.11 (m,2H), 4.08-4.00 (m, 2H), 3.87 (dd, J=10.9, 6.9 Hz, 1H), 3.42 (t, J=7.0Hz, 2H), 2.45 (t, J=7.1 Hz, 2H), 1.69-1.62 (m, 2H), 1.56-1.54 (m, 2H),1.54 (s, 9H), 1.35-1.28 (m, 2H).

Trifluoroacetic acid (3.9 mL) and H₂O (0.1 mL) were added to a solutionof 53c (66 mg, 0.125 mmol) in DCM (4 mL) at 0° C. The mixture wasstirred at 0° C. for 1 h 20 min, then ice and H₂O were added. Themixture was basified to pH 8 with saturated aqueous NaHCO₃ at 0° C. Theorganic and aqueous layers were separated, the organic layer washed withH₂O (1×), dried (Na₂SO₄) and solvent removed under vacuum to give(R)—N-(1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide53d (50 mg, 94%) as a yellow solid which was used in the next stepwithout purification. ¹H NMR δ (400 MHz, DMSO-d₆) 9.70 (s, 1H), 7.87 (d,J=8.5 Hz, 1H), 7.64 (d, J=8.2 Hz, 1H), 7.39 (ddd, J=8.1, 6.9, 1.0 Hz,1H), 7.21-7.15 (m, 2H), 7.01 (s, 2H), 5.92 (s, 1H), 4.02-3.96 (m, 1H),3.85 (dd, J=10.8, 3.5 Hz, 1H), 3.69 (t, J=9.3 Hz, 1H), 3.63-3.55 (m,2H), 3.42 (t, J=7.0 Hz, 2H), 2.42 (t, J=7.1 Hz, 2H), 1.64 (td, J=15.2,7.6 Hz, 2H), 1.55 (td, J=14.5, 7.2 Hz, 2H), 1.35-1.26 (m, 2H).

A mixture of 53d (50 mg, 0.117 mmol),(R)-5-(1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 53e (56 mg, 0.161 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI.HCl (58mg, 0.303 mmol) and TsOH (8 mg, 0.0465 mmol) in dry DMA (2 mL) wasstirred at r.t. overnight, under nitrogen. After 19 h the mixture wasdiluted with H₂O and a solid precipitated out of solution. The aqueoussuspension was extracted with EtOAc (1×), DCM (1×), DCM:MeOH 95:5 (1×)and the combined organics dried (Na₂SO₄) and solvents removed undervacuum. The crude product was purified by column chromatography onsilica gel using DCM:MeOH 100:0 to 94:6 to giveN—((S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide53 (15 mg, 17%, HPLC purity: 82.3%) as a yellow solid. ¹H NMR δ (400MHz, DMSO-d₆) 10.35 (s, 1H), 9.88 (s, 1H), 8.58 (s, 1H), 8.08 (d, J=8.2Hz, 1H), 8.02 (s, 1H), 7.96 (d, J=8.3 Hz, 1H), 7.92 (d, J=8.4 Hz, 1H),7.78 (d, J=8.3 Hz, 1H), 7.55 (t, J=7.4 Hz, 1H), 7.51-7.47 (m, 1H),7.46-7.42 (m, 1H), 7.33-7.30 (m, 1H), 7.01 (s, 2H), 4.42-4.28 (m, 3H),4.25-4.13 (m, 3H), 4.01 (ddd, J=19.6, 10.9, 2.6 Hz, 2H), 3.90 (dd,J=10.9, 7.5 Hz, 1H), 3.79 (dd, J=10.8, 8.1 Hz, 1H), 3.43 (t, J=6.7 Hz,2H), 2.77-2.57 (m, 4H), 2.46-2.43 (m, 2H), 2.01-1.96 (m, 2H), 1.70-1.62(m, 2H), 1.60-1.53 (m, 2H), 1.36-1.28 (m, 2H). HRMS m/z 777.2186[(M+Na)⁺ calcd. for C₄₁H₄₀Cl₂N₄NaO₆ 777.2217].

Example 3a1-((S)-5-amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53j

10% Pd/C (1.5 g) was added to a stirred solution of(S)-5-(5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57c (2.00 g, 4.57 mmol) in THF-25% aqueous NH₄HCO₂ (60 mL/23 mL) at−20° C., under nitrogen (FIG. 3). The reaction mixture was stirred at−15 to −10° C. for 3.5 hrs. The reaction mixture was then kept at −20°C. overnight. After 17.5 h the mixture was warmed to −10° C. and stirredat −10 to −5° C. for 5 h. The mixture was then allowed to warm to 0° C.and stirred at this temperature for 30 mins, then diluted with MeOH,filtered through celite, the celite plug washed with MeOH (3×) and thesolvents concentrated under vacuum until a solid precipitated out ofsolution. This was then diluted with H₂O (150 mL) and hexanes (150 mL)and stirred at r.t. while being acidified to pH 1 with concentrated HCl.The mixture was stirred for a further 30 min and the solid thencollected by filtration and washed with H₂O and hexanes and dried togive(S)-5-(1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 53h (1.43 g, 90%) as a beige solid. ¹H NMR δ (400 MHz, DMSO-d₆)12.07 (br s, 1H), 10.35 (s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.98 (s, 1H),7.77 (d, J=8.3 Hz, 1H), 7.50-7.46 (m, 1H), 7.33-7.29 (m, 1H), 4.30 (t,J=10.4 Hz, 1H), 4.14-4.12 (m, 2H), 3.98 (dd, J=10.9, 2.8 Hz, 1H), 3.78(dd, J=10.8, 7.8 Hz, 1H), 2.63-2.45 (m, 2H), 2.35 (t, J=7.4 Hz, 2H),1.89-1.78 (m, 2H).

Hydrogen chloride gas (HCl) was bubbled through a solution of(S)-tert-butyl1-(chloromethyl)-5-(diphenylmethyleneamino)-1H-benzo[e]indole-3(2H)-carboxylate53f (425 mg, 0.855 mmol) in dry dioxane (12 mL) (over 3 A molecularsieves), at r.t. (FIG. 3). A solid precipitated out of solution and thesolvent was removed under vacuum after 15 min. The crude solid,(S)-1-(chloromethyl)-N-(diphenylmethylene)-2,3-dihydro-1H-benzo[e]indol-5-amine53g was used in the next step without purification. Dry DMA (10 mL) wasadded to a mixture of 53g,(S)-5-(1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 53h (327 mg, 0.941 mmol), EDCI.HCl (573 mg, 2.99 mmol) and 3A(angstrom) molecular sieves at r.t., under nitrogen. After 2 days and19.5 h the solvent was removed under vacuum. The crude material waspurified by column chromatography on silica gel using DCM:MeOH 100:0 to90:10, and the material then chromatographed again using hexanes:DCM100:0 to 50:50 to 0:100, then DCM:MeOH 99:1 to 98:2 to give1-((S)-1-(chloromethyl)-5-(diphenylmethyleneamino)-1H-benzo[e]indol-3(2H)-yl)-5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53i (193 mg, 31% over 2 steps from 53f). ¹H NMR δ (400 MHz, DMSO-d₆)10.35 (s, 1H), 8.08 (d, J=8.1 Hz, 1H), 8.00 (s, 1H), 7.84 (t, J=9.0 Hz,2H), 7.79-7.74 (m, 3H), 7.62-7.57 (m, 2H), 7.54-7.46 (m, 4H), 7.40-7.36(m, 1H), 7.33-7.29 (m, 1H), 7.27-7.22 (m, 3H), 7.09-7.08 (m, 2H),4.34-4.26 (m, 2H), 4.22-4.12 (m, 4H), 4.01-3.97 (m, 2H), 3.83-3.76 (m,2H), 2.69-2.50 (m, 4H), 1.93-1.86 (m, 2H). HRMS m/z 726.2264 [(M+H)⁺calcd for C₄₄H₃₈Cl₂N₃O₃ 726.2285].

Acetic acid (HOAc, 8 mL) was added to a stirred solution of 53i (190 mg,0.261 mmol) in THF-H₂O (24 mL/12 mL) at r.t. and the mixture stirredovernight. After 19 h the mixture was diluted with H₂O and a solidprecipitated out. The THF was removed under vacuum and the aqueoussuspension treated with DIPEA until neutral. The solid was collected byfiltration, washed with H₂O and dried. The solid was dissolved in DMF(1.5 mL) and diluted with MeOH causing a solid to precipitate. Thesolvents were decanted and hexanes:DCM 90:10 were added to the solid,the suspension was shaken and the solvents decanted. This process wasrepeated using hexanes:EtOAc 90:10, followed by hexanes alone. The solidwas then dissolved in DMF/THF, absorbed onto silica gel and the producteluted using DCM:MeOH 100:0 to 90:10. The material was further purifiedby preparative HPLC (column: Synergi-MAX RP 4μ, 21.20×250 mm; flow rate:13 mL/min; mobile phase: solvent A: H₂O/TFA pH 2.5, solvent B: MeCN/H₂O90:10; method: isocratic, solvent A:solvent B 20:80, 15 min; wavelength:254 nm, 330 nm) to give1-((S)-5-amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53j, 30 mg, 22%, HPLC purity: 87.9%) as a white solid. ¹H NMR δ (400MHz, DMSO-d₆) 10.36 (s, 1H), 8.08 (d, J=8.1 Hz, 1H), 8.04-8.02 (m, 2H),7.79-7.77 (m, 2H), 7.69 (d, J=8.3 Hz, 1H), 7.51-7.47 (m, 1H), 7.42 (t,J=7.5 Hz, 1H), 7.34-7.30 (m, 1H), 7.23 (t, J=7.6 Hz, 1H), 5.91 (s, 2H),4.36-4.26 (m, 2H), 4.19-4.13 (m, 3H), 4.08-4.04 (m, 1H), 4.01-3.93 (m,2H), 3.79 (dd, J=10.7, 8.2 Hz, 1H), 3.70 (dd, J=10.5, 8.9 Hz, 1H),2.75-2.66 (m, 2H), 2.63-2.54 (m, 2H), 2.00-1.93 (m, 2H). HRMS m/z584.1456 [(M+Na)⁺ calcd for C₃₁H₂₉Cl₂N₃NaO₃ 584.1478]. [t]D²⁸=−37.6°(c=0.559, DMSO).

Example 3b 1-((R)-5-amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-((R)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53p

Triethylamine (Et₃N, 0.54 mL, 3.89 mmol) and triflic anhydride (0.60 mL,3.60 mmol) were added to a stirred solution of (R)-tert-butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 53k (1.00g, 3.00 mmol) in DCM (100 mL) at 0° C. (FIG. 4). The reaction wasstirred at 0° C. for 20 min, then diluted with H₂O, the layers wereseparated and the aqueous layer was extracted with DCM (1×). Thecombined organic layers were dried (Na₂SO₄) and solvent was removedunder vacuum. Purification by column chromatography on silica gel usinghexanes:EtOAc 100:0 to 96:4 gave (R)-tert-butyl1-(chloromethyl)-5-(trifluoromethylsulfonyloxy)-1H-benzo[e]indole-3(2H)-carboxylate53l (1.30 g, 93%) as an orange foamy solid. ¹H NMR δ (400 MHz, CDCl₃)8.30 (br s, 1H), 8.03 (d, J=8.5 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H),7.62-7.58 (m, 1H), 7.53-7.49 (m, 1H), 4.32 (br s, 1H), 4.20-4.15 (m,1H), 4.09-4.03 (m, 1H), 3.92 (dd, J=11.2, 2.8 Hz, 1H), 3.54-3.49 (m,1H), 1.61 (s, 9H).

A solution of 53l (1.30 g, 2.79 mmol) in dry THF (60 mL, degassed) wasadded to a mixture of Cs₂CO₃ (1.27 g, 3.91 mmol), BINAP (209 mg, 0.336mmol) and Pd(OAc)₂ (63 mg, 0.281 mmol), under nitrogen.Diphenylmethanimine (0.56 mL, 3.34 mmol) was then added and the mixturerefluxed overnight, under nitrogen. After 20 h the reaction temperaturewas reduced to 60-65° C. and the reaction was stirred at thistemperature under nitrogen for 1 day. Additional THF (10 mL) was addedand the mixture stirred at the same temperature for another day beforemore THF (25 mL) was again added to the mixture. After another dayadditional portions of Pd(OAc)₂ (19 mg, 0.0846 mmol), BINAP (52 mg,0.0835 mmol) and THF (30 mL) were added and the mixture heated at 70° C.overnight, under nitrogen. After a further 28 h additional portions ofPd(OAc)₂ (31 mg, 0.138 mmol), BINAP (104 mg, 0.167 mmol) were againadded and the reaction continued for 22 h. The reaction mixture was thencooled to r.t., diluted with DCM, filtered through celite, the celiteplug washed with DCM until there was no more color in the washings andthe filtrate evaporated under vacuum. Purification by columnchromatography on silica gel using hexanes:DCM 100:0 to 50:50 gave(R)-tert-butyl1-(chloromethyl)-5-(diphenylmethyleneamino)-1H-benzo[e]indole-3(2H)-carboxylate53a (1.19 g, 85%) as a yellow, foamy solid. ¹H NMR δ (400 MHz, DMSO-d₆)7.85 (d, J=8.3 Hz, 1H), 7.80 (d, J=8.4 Hz, 1H), 7.75 (d, J=7.2 Hz, 2H),7.61-7.57 (m, 1H), 7.54-7.49 (m, 3H), 7.37-7.33 (m, 1H), 7.30-7.23 (m,3H), 7.06 (d, J=6.8 Hz, 2H), 4.13-4.02 (m, 2H), 4.00-3.94 (m, 2H), 3.77(dd, J=10.9, 7.5 Hz, 1H), 1.46 (s, 9H), 1H not observed. [α]_(D)²⁷=+101° (c=1.04, DCM).

Hydrogen chloride gas (HCl (g)) was bubbled through a solution of 53a(300 mg, 0.604 mmol) in dry dioxane (10 mL) (over 3 A molecular sieves)at r.t. After 10 min a solid had precipitated out of solution and thesolvent was removed under vacuum after 20 mins. The crude solid,(R)-1-(chloromethyl)-N-(diphenylmethylene)-2,3-dihydro-1H-benzo[e]indol-5-amine53m was used in the next step without purification.

Palladium on carbon, 10% Pd/C (690 mg) was added to a stirred solutionof(R)-5-(5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 53n (1.38 g, 3.15 mmol) in THF-25% aqueous NH₄HCO₂ (40 mL/16 mL) at−10° C., under nitrogen. The reaction mixture was stirred at −10 to −5°C. for 3 h. The reaction mixture was then kept at −20° C. overnight.After 15 h at −20° C. the mixture was diluted with MeOH, filteredthrough celite, the celite plug washed with MeOH and the solventsconcentrated under vacuum until a solid precipitated out of solution.The suspension was then diluted with H₂O (130 mL) and hexanes (100 mL)and stirred at r.t. while being acidified to pH 1 with concentrated HCl.The mixture was stirred for 30 mins, let settle and the hexanesdecanted. Additional hexanes (120 mL) was added and the mixture stirredfor another 30 mins, the hexanes decanted again and the solid thencollected by filtration and washed with H₂O and hexanes and dried togive(R)-5-(1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 53e (925 mg, 84%) as a beige solid. ¹H NMR δ (400 MHz, DMSO-d₆)12.06 (br s, 1H), 10.35 (s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.98 (s, 1H),7.77 (d, J=8.3 Hz, 1H), 7.50-7.46 (m, 1H), 7.33-7.29 (m, 1H), 4.30 (t,J=10.5 Hz, 1H), 4.14-4.12 (m, 2H), 3.98 (dd, J=10.9, 2.8 Hz, 1H), 3.78(dd, J=10.8, 7.9 Hz, 1H), 2.63-2.45 (m, 2H), 2.35 (t, J=7.4 Hz, 2H),1.89-1.78 (m, 2H).

Dry DMA (8 mL) was added to a mixture of 53m from the previous reaction,53e (241 mg, 0.693 mmol), EDCI.HCl (404 mg, 2.11 mmol) and 3A molecularsieves at r.t., under nitrogen. The reaction mixture was stirredovernight. After 19.5 h the mixture was diluted with H₂O and theresulting suspension filtered. The aqueous filtrate was extracted withEtOAc (3×) and the filtered solid dissolved in EtOAc/MeOH and combinedwith the EtOAc extracts. The combined organic solution was absorbed ontosilica gel and the product eluted using hexanes:DCM 50:50 to 0:100, thenDCM:MeOH 99.5:0.5 to 97:3 to give1-((R)-1-(chloromethyl)-5-(diphenylmethyleneamino)-1H-benzo[e]indol-3(2H)-yl)-5-((R)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53o (155 mg, 35% over 2 steps from 53a) as a dark yellow solid. ¹H NMR δ(400 MHz, DMSO-d₆) 10.35 (s, 1H), 8.08 (d, J=8.0 Hz, 1H), 8.00 (s, 1H),7.85 (t, J=8.9 Hz, 2H), 7.79-7.74 (m, 3H), 7.62-7.57 (m, 2H), 7.54-7.46(m, 4H), 7.40-7.36 (m, 1H), 7.33-7.29 (m, 1H), 7.27-7.22 (m, 3H),7.09-7.08 (m, 2H), 4.34-4.27 (m, 2H), 4.22-4.11 (m, 4H), 4.02-3.97 (m,2H), 3.83-3.76 (m, 2H), 2.69-2.51 (m, 4H), 1.93-1.85 (m, 2H), NMRspectrum matches that of 53i. HRMS m/z 748.2077 [(M+Na)⁺ calcd forC₄₄H₃₇Cl₂N₃NaO₃ 748.2104].

Acetic acid (HOAc, 4 mL) was added to a stirred solution of 53o (80 mg,0.110 mmol) in THF-H₂O (12 mL/6 mL) at r.t. and the mixture stirredovernight. After 18 h the mixture was diluted with H₂O and a solidprecipitated out. The THF was removed under vacuum and the aqueoussuspension treated with DIPEA until pH 8, resulting in the precipitationof more solid. The solid was collected by filtration, dried, andpurified by column chromatography on silica gel using DCM:MeOH 100:0 to97:3 to give1-((R)-5-amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-((R)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione53p (20 mg, 32%) as a tan solid. ¹H NMR δ (400 MHz, DMSO-d₆) 10.36 (s,1H), 8.08 (d, J=8.2 Hz, 1H), 8.04-8.02 (m, 2H), 7.79-7.77 (m, 2H), 7.69(d, J=8.3 Hz, 1H), 7.51-7.47 (m, 1H), 7.42 (t, J=7.5 Hz, 1H), 7.34-7.30(m, 1H), 7.23 (t, J=7.6 Hz, 1H), 5.91 (s, 2H), 4.36-4.26 (m, 2H),4.19-4.13 (m, 3H), 4.08-4.04 (m, 1H), 4.01-3.93 (m, 2H), 3.79 (dd,J=10.7, 8.3 Hz, 1H), 3.71 (dd, J=10.5, 8.9 Hz, 1H), 2.75-2.66 (m, 2H),2.63-2.53 (m, 2H), 2.00-1.93 (m, 2H), NMR spectrum matches that of 53j.

Example 4N-(4-(((S)-1-(chloromethyl)-3-(6-((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)hexanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide54

A mixture of(S)-(2-amino-4-hydroxy-5-methoxyphenyl)(2-(hydroxymethyl)pyrrolidin-1-yl)methanone54a (7.6 g, 28.6 mmol), prepared by the procedures of Tercel et al(2003) J. Med. Chem 46:2132-2151, and di-t-butyl dicarbonate (12.48 g,57.2 mmol) in anhydrous THF (140 mL) was stirred under reflux in anitrogen atmosphere for 18 h. The reaction mixture was cooled to r.t.and 2N NaOH (57.2 mL, 114 mmol) and MeOH (70 mL) were added. The mixturewas stirred at r.t. for 6 h. Volatiles were evaporated under reducedpressure at 35-40° C. (bath temperature). Ice water (250 mL) was addedand the pH was adjusted to 8-9 at 0° C. The mixture was stirred withpetroleum ether-ethyl acetate (20:1) (2×400 mL) at r.t. for 15 min. Theorganic layer was separated and discarded. The aqueous layer wasextracted with DCM (4×300 mL) and the combined extracts were dried(MgSO₄) and evaporated under reduced pressure to give (S)-tert-butyl5-hydroxy-2-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxyphenylcarbamate54b as a pink-white solid (9.36 g, 89%); mp 154-156° C.; ¹H NMR[(CD₃)₂SO] δ 9.51 (s, 1H), 8.90 (s, 1H), 7.27 (s, 1H), 6.91 (s, 1H),4.73 (t, J=5.8 Hz, 1H), 4.16-4.02 (m, 1H), 3.73 (s, 3H), 3.64-3.34 (m,4H), 1.99-1.60 (m, 4H), 1.43 (s, 9H). Anal. (C₁₈H₂₆N₂O₆) Calc: C, 59.00;H, 7.15; N, 7.65. Found: C, 58.94; H, 7.31; N, 7.39.

To a solution of 54b (2.88 g, 7.87 mmol) and 2,2,2-trichloroethyl6-bromohexanoate (3.86 g, 11.8 mmol), prepared by the procedures inTercel et al (2003) J. Med. Chem 46:2132-2151, in dry DMA (7 mL) wasadded anhydrous K₂CO₃ (2.61 g, 18.9 mmol). The resulting mixture wasstirred at r.t. for 68 h. It was poured into ice-water (600 mL) and theproduct was extracted into ethyl acetate (600 mL). The extracts werewashed successively with cold (0° C.) aqueous 2N Na₂CO₃ solution (2×400mL) and water (400 mL) and then dried (MgSO₄). Evaporation of thesolvent gave a brown oil which was purified by SiO₂ columnchromatography (DCM-ethyl acetate=2:1) to give pure(S)-2,2,2-trichloroethyl6-(5-(tert-butoxycarbonylamino)-4-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)hexanoate54c (3.62 g, 76%) as a pale yellow foam; mp 36-39° C.; ¹H NMR [(CD₃)₂SO]δ 9.90 (s, 1H), 7.33 (s, 1H), 6.93 (s, 1H), 4.89 (s, 2H), 4.74 (t, J=5.8Hz, 1H), 4.17-4.02 (m, 1H), 3.94 (t, J=6.4 Hz, 2H), 3.73 (s, 3H),3.63-3.26 (m, 4H), 2.55-2.46 (m, 2H, partially obscured by DMSO peak),2.00-1.55 (m, 8H), 1.53-1.36 (m, 11H). Anal. (C₂₆H₃₇N₂O₈) Calc: C,51.03; H, 6.09; N, 4.58. Found: C, 51.33; H, 6.21; N, 4.35.

To a solution of 54c (3.62 g, 5.92 mmol) in dry DCM (12 mL) was addedDess-Martin periodinane (DMP,1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one, CAS Reg. No.87413-09-0) (3.27 g, 7.70 mmol) portionwise over 15 min at r.t. Thereaction mixture was stirred at r.t. for 45 min. It was diluted with DCM(800 mL) and washed successively with 10% Na₂S₂O₃ (100 mL), cold (0° C.)NaHCO₃ solution (400 mL), and water (300 mL) and then dried (MgSO₄).Evaporation of the solvent gave an amber solid which was purified bySiO₂ column chromatography (petroleum ether-ethyl acetate=3:4) to give(S)-tert-butyl11-hydroxy-7-methoxy-5-oxo-8-(6-oxo-6-(2,2,2-trichloroethoxy)hexyloxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate54d (2.61g, 72%) as a sticky foam; ¹H NMR [(CD₃)₂SO] δ 7.03 (s, 1H),6.67 (s, 1H), 6.38 (s, 1H), 5.41 (s, 1H), 4.89 (s, 2H), 4.06-3.87 (m,2H), 3.79 (s, 3H), 3.52-3.43 (m, 1H), 3.42-3.28 (m, 1H, partiallyobscured by water peak), 3.27-3.20 (m, 1H), 2.08-1.82 (m, 5H), 1.81-1.71(m, 2H), 1.71-1.61 (m, 2H), 1.53-1.40 (m, 3H), 1.31 (s, 9H). HRMS (ESI)m/z calc. for C₂₆H₃₅Cl₃N₂NaO₈: 631.1351. found: 631.1361 [MNa⁺].

To a stirred solution of 54d (1.80 g, 2.95 mmol) in acetone-water (3:2)(100 mL) under nitrogen was added Zn (7.72 g, 118 mmol) and NH₄Cl (6.32g, 118 mmol). The mixture was stirred at r.t. for 28 h. The supernatantwas decanted and the Zn residue was washed with aqueous NaHCO₃ (3×100mL). The washes and the supernatant were combined and stirred with DCM(300 mL then 2×100 mL). The DCM layers were separated and discarded. Theaqueous layer was acidified with conc. HCl to pH<1 at 0° C. The productwas extracted into DCM (400 mL; 2×200 mL) which was dried (MgSO₄) andevaporated to give (S)-tert-butyl8-(5-hydroperoxyhex-5-enyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate54e (1.34 g, 95%) as a colorless foam; mp 65-67° C.; ¹H NMR [(CD₃)₂SO] δ11.99 (br s, 1H), 7.04 (s, 1H), 6.67 (s, 1H), 6.37 (br s, 1H), 5.41 (d,J=9.4 Hz, 1H), 4.06-3.87 (m, 2H), 3.79 (s, 3H), 3.53-3.19 (m, 3H,partially obscured by water peak), 2.22 (t, J=7.2 Hz, 2H), 2.09-1.81 (m,4H), 1.80-1.67 (m, 2H), 1.62-1.49 (m, 2H), 1.49-1.37 (m, 2H), 1.31 (s,9H). Anal. (C₂₄H₃₄N₂O₈. ¼H₂O) Calc: C, 59.68; H, 7.20; N, 5.80. Found:C, 59.37; H, 7.20; N, 5.62.

A mixture of 54e (1.16 g, 2.42 mmol),(S)-1-(chloromethyl)-5-(4-nitrobenzyloxy)-2,3-dihydro-1H-benzo[e]indole54f (893 mg, 2.42 mmol), EDCI.HCl (1.39 g, 7.26 mmol), and anhydrousTsOH (83 mg, 0.48 mmol) in DMA (7 mL) was stirred at room temperatureunder a nitrogen atmosphere for 4 h. Water (120 mL) was added and themixture was stirred at r.t. for 15 min. The precipitated solid wasfiltered off, washed successively with water (4×40 mL), 0.01% NH₄OH(4×40 mL), and petroleum ether (4×40 mL) and then dried to giveN-Boc-(S)-8-(6-((S)-1-(chloromethyl)-5-(4-nitrobenzyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(10H)-one54g (1.71 g, 85%) as a pale yellow solid; mp 130-133° C.; ¹H NMR[(CD₃)₂SO] δ 8.30 (d, J=8.8 Hz, 2H), 8.23 (d, J=8.1 Hz, 1H), 8.18 (s,1H), 7.92-7.82 (m, 3H), 7.57 (td, J=8.2, 1.1 Hz, 1H), 7.43 (t, J=8.0 Hz,1H), 7.03 (s, 1H), 6.69 (s, 1H), 6.39 (br s, 1H), 5.51-5.35 (m, 3H),4.36 (t, J=9.5 Hz, 1H), 4.28-4.15 (m, 2H), 4.10-3.90 (m, 3H), 3.85 (dd,J=11.1, 7.8 Hz, 1H), 3.79 (s, 3H), 3.52-3.42 (m, 1H), 3.42-3.20 (m, 2H,partially obscured by water peak), 2.68-2.50 (m, 2H, partially obscuredby DMSO peak), 2.11-1.95 (m, 1H), 1.95-1.75 (m, 5H), 1.75-1.61 (m, 2H),1.59-1.45 (m, 2H), 1.31 (s, 9H). Anal. (C₄₄H₄₉ClN₄O₁₀) Calc: C, 63.72;H, 5.96; N, 6.76. Found: C, 63.33; H, 5.97; N, 6.92.

To a stirred solution 54g (1.70 g, 2.05 mmol) in a mixture of THF (90mL), acetone (70 mL), and water (40 mL) under nitrogen was added Zn(2.68 g, 41.0 mmol) and NH₄Cl (4.39 g, 82.0 mmol). The mixture wasstirred at r.t. for 45 min, then filtered through celite, washing withTHF several times. The filtrate was concentrated under reduced pressureat r.t. to ca. 60 mL. A solution of 0.01% NH₄OH (400 mL) was added andthe mixture was stirred at r.t. for 15 min. The solid was collected andwashed successively with 0.01% NH₄OH (3×100 mL), water (3×100 mL), andpetroleum ether (3×100 mL). The solid was dried to give the anilinoderivative of 54g (1.16 g, 100%) which was treated with6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (253 mg, 1.20mmol), EDCI.HCl (576 mg, 3.00 mmol) and anhydrous TsOH (34.4 mg, 0.20mmol) in dry DMA (2 mL). The mixture was stirred at r.t. and undernitrogen for 17 h. NaHCO₃ solution (50 mL) was added and the mixture wasstirred at r.t. for 30 min. The solid was collected, washed with water,dried, and purified by a silica column chromatography (DCM-ethylacetate=1:1) to give pure (S)-tert-butyl8-(6-((S)-1-(chloromethyl)-5-(4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate54h (194 mg, 33%) as a yellow solid, mp 128-132° C.; ¹H NMR [(CD₃)₂SO] δ9.91 (s, 1H), 8.20-8.11 (m, 2H), 7.84 (d, J=8.3 Hz, 1H), 7.62 (d, J=8.5Hz, 2H), 7.54 (br t, J=8.1 Hz, 1H), 7.47 (d, J=8.5 Hz, 2H), 7.38 (br t,J=8.0 Hz, 1H), 7.04 (s, 1H), 7.00 (s, 2H), 6.69 (s, 1H), 6.38 (br s,1H), 5.45-5.37 (m, 1H), 5.20 (s, 2H), 4.36 (t, J=10.2 Hz, 1H), 4.26-4.13(m, 2H), 4.10-3.92 (m, 3H), 3.88-3.79 (m, 1H), 3.79 (s, 3H), 3.52-3.30(m, 4H), 3.24 (br t, J=8.9 Hz, 1H), 2.66-2.50 (m, 2H, partially obscuredby DMSO peak), 2.28 (t, J=7.3 Hz, 2H), 2.08-1.95 (m, 1H), 1.95-1.76 (m,5H), 1.76-1.64 (m, 2H), 1.64-1.20 (m, 8H), 1.31 (s, 9H). Anal.(C₅₄H₆₂ClN₅O₁₁) Calc: C, 65.35; H, 6.30; N, 7.06. Found: C, 65.08; H,6.39; N, 6.67.

To a solution ofN-(4-(((S)-1-(chloromethyl)-3-(6-((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)hexanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide54h (204 mg, 0.21 mmol) in DCM (15 mL) stirred at −10 to −11° C. under anitrogen atmosphere was added dropwise (over 30 min) TFA containing 2.5%water (15 mL). After addition, the mixture was stirred further at thistemperature for 4 h. The mixture was poured into a mixture of ice, DCM,and sufficient saturated NaHCO₃ solution to give a pH 7-8 at 0° C. Themixture was stirred at r.t. for 15 min. The DCM layer was separated andwashed with more aqueous NaHCO₃ and water and then dried (MgSO₄). Thesolvent was evaporated at 25° C. (bath temperature) to give a yellowsolid (172 mg, 94% material recovered). This crude product was purifiedby preparative HPLC (Synergi-Max RP column) (eluted with 30% ammoniumformate buffer pH=3.5; 70% aqueous (10%) acetonitrile; flow rate: 13mL/min) to give 54 (30 mg, 16%), HPLC: 98.8% pure; mp 190° C. (dec.);[α²⁰ _(D)+320° (c 0.100, DCM); ¹H NMR [CDCl₃] δ 8.29 (d, J=8.3 Hz, 1H),8.18 (s, 1H), 7.69-7.63 (m, 2H), 7.61-7.44 (m, 6H), 7.37 (br t, J=7.3Hz, 2H), 6.82 (s, 1H), 6.66 (s, 2H), 5.24 (s, 2H), 4.34-4.19 (m, 2H),4.19-4.00 (m, 3H), 3.99-3.92 (m, 1H), 3.89 (s, 3H), 3.86-3.78 (m, 1H),3.76-3.70 (m, 1H), 3.63-3.49 (m, 3H), 3.42 (t, J=10.8 Hz, 1H), 2.68-2.47(m, 2H), 2.40-2.27 (m, 3H), 2.12-1.92 (m, 5H), 1.92-1.82 (m, 2H),1.82-1.71 (m, 2H), 1.71-1.54 (m, 4H, partially obscured by water peak),1.43-1.32 (m, 2H). HRMS (ESI) m/z calc. for C₄₉H₅₂ClN₅NaO₈: 896.3397.found: 896.3375 [MNa⁺].

Example 5N—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide55

Potassium carbonate, K₂CO₃ (2.50 g, 18.1 mmol) was added to a mixture of(S)-tert-butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a (2.01g, 6.02 mmol) and 1-(bromomethyl)-4-nitrobenzene (5.20 g, 24.1 mmol) inDMF (12 mL) at room temperature (FIG. 6). The reaction mixture wasstirred at r.t. for 2 h, then diluted with EtOAc and H₂O and the layersseparated. The organic layer was washed with H₂O (3×), brine (1×), dried(Na₂SO₄) and solvent removed under vacuum. Purification by columnchromatography on silica gel twice using hexanes:EtOAc 100:0 to 96:4gave (S)-tert-butyl1-(chloromethyl)-5-(4-nitrobenzyloxy)-1H-benzo[e]indole-3(2H)-carboxylate55a (2.17 g, 77%) as a bright yellow solid. ¹H NMR δ (400 MHz, CDCl₃)8.31-8.27 (m, 3H), 7.85 (br s, 1H), 7.72 (d, J=8.4 Hz, 2H), 7.67 (d,J=8.3 Hz, 1H), 7.54 (ddd, J=8.2, 6.8, 1.2 Hz, 1H), 7.38 (ddd, J=8.2,6.8, 1.1 Hz, 1H), 4.28-4.25 (m, 1H), 4.16-4.10 (m, 1H), 4.02-3.92 (m,2H), 3.45 (t, J=10.6 Hz, 1H), 1.60 (s, 9H). HRMS m/z 491.1338 [(M+Na)⁺calcd for C₂₅H₂₅ClN₂NaO₅ 491.1344].

Reduction Method A: 55a (1.53 g, 3.26 mmol) was dissolved in THF-acetone(75 mL/60 mL). H₂O (30 mL) was added once 55a had dissolved. NH₄Cl (10.5g, 196 mmol) and Zn powder (6.40 g, 97.9 mmol) were added and theresulting mixture stirred at r.t., under nitrogen for 1 h. The reactionmixture was then filtered through celite, the celite plug washed withDCM and the combined filtrates washed with H₂O (1×), dried (Na₂SO₄) andsolvent removed under vacuum to give compound 55b as an orange solid.The crude product was used in the next step without purification.

Reduction Method B: Mercury-aluminium amalgam was added to a solution of55a in THF-MeOH—H₂O (150 mL/50 mL/20 mL). After 15 min the reactionmixture was diluted with DCM, filtered through celite and the celiteplug washed with DCM. The organics were washed with H₂O, dried (Na₂SO₄)and solvents removed under vacuum to give (S)-tert-butyl5-(4-aminobenzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate55b as an orange solid. The product was used in the next step withoutpurification. ¹H NMR δ (400 MHz, DMSO-d₆) 8.06 (d, J=8.6 Hz, 1H), 7.80(d, J=8.3 Hz, 1H), 7.53-7.49 (m, 1H), 7.34-7.30 (m, 1H), 7.19 (d, J=8.2Hz, 2H), 6.60 (d, J=8.4 Hz, 2H), 5.16 (s, 2H), 5.04 (d, J=1.3 Hz, 2H),4.18-4.05 (m, 3H), 4.00-3.97 (m, 1H), 3.81 (dd, J=10.9, 6.9 Hz, 1H),1.56 (s, 9H), 1H not observed.

A mixture of(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid(Fmoc-L-citrulline, 3.26 g, 8.20 mmol) and2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, CAS Reg. No.16357-59-8, 3.12 g, 12.6 mmol) in DMA (15 mL) was stirred at r.t., undernitrogen for 20 min. A solution of 55b (2.77 g, 6.31 mmol) in DMA (15mL) was then added, the resulting mixture flushed with nitrogen and leftstirring overnight. After 16 h the reaction mixture was poured over iceand diluted with H₂O. The resulting precipitate was filtered off, washedwith H₂O, dissolved in DCM/MeOH, dried (Na₂SO₄) and solvents removedunder vacuum. The crude product was purified by trituration where theproduct was precipitated with hexanes:EtOAc 94:6, the solvents decantedand the process repeated using hexanes:EtOAc 90:10 and thenhexanes:EtOAc 95:5. The material was then columned on silica gel usingDCM:MeOH 100:0 to 95:5 to give (S)-tert-butyl5-(4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate55c (4.23 g, 79% over two steps from 55a, HPLC purity: 95.3%) as ayellow solid. ¹H NMR δ (400 MHz, DMSO-d₆) 10.12 (s, 1H), 8.12 (d, J=8.4Hz, 1H), 7.89 (d, J=7.5 Hz, 2H), 7.82 (d, J=8.3 Hz, 1H), 7.77-7.74 (m,2H), 7.70-7.67 (m, 3H), 7.55-7.49 (m, 3H), 7.42 (t, J=7.4 Hz, 2H),7.37-7.31 (m, 3H), 6.00 (t, J=5.7 Hz, 1H), 5.43 (s, 2H), 5.22 (s, 2H),4.29-4.27 (m, 2H), 4.24-4.05 (m, 6H), 4.01-3.98 (m, 1H), 3.82 (dd,J=10.9, 7.0 Hz, 1H), 3.09-2.92 (m, 2H), 1.74-1.35 (m, 4H), 1.55 (s, 9H).HRMS m/z 840.3101 [(M+Na)⁺ calcd for C₄₆H₄₈ClN₅NaO₇ 840.3134].

Piperidine (1.5 mL, 10% v/v) was added to a stirred solution of 55c(4.18 g, 5.11 mmol) in DMF (15 mL) at r.t. The reaction mixture wasstirred for 1 h. The resulting suspension was diluted with hexanes:EtOAc90:10 (100 mL) and stirred for 10 min. Two layers formed and the toplayer was decanted off. The solvent in the retained bottom layer wasremoved under vacuum. Purification by column chromatography on silicagel using DCM:MeOH 100:0 to 85:15 gave (S)-tert-butyl5-(4-((S)-2-amino-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate55d (2.97 g, 97%, HPLC purity: 99.1%) as a yellow powder. ¹H NMR δ (400MHz, DMSO-d₆) 9.93 (br s, 1H), 8.12 (d, J=8.3 Hz, 1H), 7.82 (d, J=8.3Hz, 1H), 7.70 (d, J=8.6 Hz, 2H), 7.55-7.48 (m, 3H), 7.37-7.33 (m, 1H),5.94 (t, J=5.7 Hz, 1H), 5.37 (s, 2H), 5.22 (s, 2H), 4.19-4.05 (m, 4H),4.01-3.98 (m, 1H), 3.82 (dd, J=10.9, 7.0 Hz, 1H), 3.04-2.91 (m, 2H),1.71-1.36 (m, 4H), 1.55 (s, 9H), 3H not observed. HRMS m/z 596.2627[(M+H)⁺ calcd for C₃₁H₃₉ClN₅O₅ 596.2634].

A mixture of (S)-2,5-dioxopyrrolidin-1-yl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanoate(Fmoc-Val-OSu, 3.19 g, 7.31 mmol) and 55d (2.91 g, 4.87 mmol) in DMA (15mL) was stirred overnight at r.t., under nitrogen. After 20 hhexanes:EtOAc 80:20 (150 mL) were added and the suspension stirred for30 min. The solvents were then decanted leaving behind a solid. This wasrepeated using hexanes:EtOAc 75:25 several times. The solid was thensuspended in DCM:MeOH 75:25 and the suspension sonicated. The suspensionwas diluted with hexanes (200 mL) and the solid filtered off and washedwith hexanes:EtOAc 65:35 and dried to give (S)-tert-butyl5-(4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate55e (3.79 g, 85%, HPLC purity: 92.4%) as an orange powder. ¹H NMR δ (400MHz, DMSO-d₆) 10.12 (s, 1H), 8.14-8.12 (m, 2H), 7.89 (d, J=7.5 Hz, 2H),7.82 (d, J=8.4 Hz, 1H), 7.75 (t, J=7.6 Hz, 2H), 7.66 (d, J=8.5 Hz, 2H),7.55-7.48 (m, 3H), 7.45-7.30 (m, 6H), 5.98 (t, J=5.7 Hz, 1H), 5.41 (s,2H), 5.22 (s, 2H), 4.45 (dd, J=13.6, 7.7 Hz, 1H), 4.36-4.04 (m, 6H),4.02-3.90 (m, 2H), 3.82 (dd, J=10.8, 6.9 Hz, 1H), 3.08-2.91 (m, 2H),2.05-1.96 (m, 1H), 1.76-1.34 (m, 4H), 1.55 (s, 9H), 0.89 (d, J=6.8 Hz,3H), 0.86 (d, J=6.8 Hz, 3H). HRMS m/z 939.3808 [(M+Na)⁺ calcd forC₅₁H₅₇ClN₆NaO₈ 939.3819].

Boc removal Method A: TFA (9.5 mL) was added portion wise to asuspension of 55e (685 mg, 0.747 mmol) in DCM (19 mL) at 0° C. Thereaction mixture was stirred at 0° C. for 1 h 45 min. Aqueous NH₃(0.25%, 100 mL) was then added portion wise to the mixture at 0° C.,followed by the addition of concentrated aqueous NH₃ until pH 9-10.Hexanes:EtOAc 90:10 were then added and the mixture stirred at 0° C. for40 mins, the suspension sonicated and the precipitate collected byfiltration, washed with H₂O, H₂O:MeOH 80:20, hexanes:EtOAc 60:40,hexanes:Et₂O 50:50, hexanes and dried to give (9H-fluoren-9-yl)methyl(S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate55f as a brown solid (364 mg, 60%).

Boc removal Method B: BF₃.Et₂O (0.07 mL, 0.552 mmol) was added to asuspension of 55e (0.106 g, 0.116 mmol) in DCM (40 mL), at 0° C. After 1h 50 mins the suspension was concentrated under vacuum at r.t. untilonly a little DCM remained. A little MeOH was added until theprecipitate dissolved and the solution was then diluted with H₂O. Asolid precipitated out and the H₂O was decanted. Hexanes:EtOAc 90:10 (20mL) were added and the suspension sonicated before the solid wascollected by filtration. The solid was washed with H₂O and hexanes anddried to give (9H-fluoren-9-yl)methyl(S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate55f (66 mg, 70%) as a brown solid. ¹H NMR δ (400 MHz, DMSO-d₆) 10.10 (s,1H), 8.12 (d, J=7.6 Hz, 1H), 8.00 (d, J=8.3 Hz, 1H), 7.89 (d, J=7.4 Hz,2H), 7.74 (t, J=7.7 Hz, 2H), 7.64 (d, J=8.5 Hz, 2H), 7.58 (d, J=8.3 Hz,1H), 7.46-7.37 (m, 5H), 7.34-7.30 (m, 2H), 7.13-7.09 (m, 1H), 6.55 (s,1H), 5.97 (t, J=5.7 Hz, 1H), 5.41 (s, 2H), 5.17 (s, 2H), 4.43 (dd,J=13.0, 7.5 Hz, 1H), 4.34-4.21 (m, 3H), 3.95-3.90 (m, 2H), 3.83 (dd,J=10.7, 3.4 Hz, 1H), 3.70-3.66 (m, 1H), 3.61-3.51 (m, 2H), 3.08-2.90 (m,2H), 2.04-1.96 (m, 1H), 1.76-1.33 (m, 4H), 0.89 (d, J=6.8 Hz, 3H), 0.86(d, J=6.8 Hz, 3H), 2H not observed. HRMS m/z 839.3266 [(M+Na)⁺ calcd forC₄₆H₄₉ClN₆NaO₆ 839.3294].

A mixture of 55f (485 mg, 0.593 mmol), 53h (206 mg, 0.593 mmol),EDCI.HCl (293 mg, 1.48 mmol) and TsOH (26 mg, 0.151 mmol) in DMA (10 mL)was flushed with nitrogen and stirred at r.t. overnight. After 19.5 hthe reaction mixture was diluted with H₂O and the resulting solidcollected by filtration, washed with H₂O and hexanes:EtOAc 50:50.Filtration column chromatography on a plug of silica gel using DCM: MeOH100:0 to 90:10 gave (9H-fluoren-9-yl)methyl(S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate55g (391 mg) used crude in the next step. HRMS m/z 1144.4169 [(M−H)⁺calcd for C₆₄H₆₄Cl₂N₇O₉ 1144.4148].

Piperidine (0.39 mL, 3.95 mmol) was added to a suspension of 55g (910mg, 0.793 mmol) in DMF (6 mL) at r.t. After 10 min the mixture wasconcentrated under vacuum. Purification by column chromatography onsilica gel using DCM:MeOH 95:5 to 85:15 followed by further purificationby preparative HPLC (column: Synergi-MAX RP 4μ, 21.20×250 mm; flow rate:12 mL/min; mobile phase: solvent A: H₂O/TFA pH 2.47, solvent B: MeCN/H₂O90:10; method: gradient, solvent A:solvent B 60:40 to 22:78 to 60:40, 24min; wavelength: 254 nm, 330 nm) gave(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(((S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenyl)-5-ureidopentanamidetrifluoroacetate 55h (75 mg, 9%) as a cream solid. ¹H NMR δ (400 MHz,DMSO-d₆) 10.35 (s, 1H), 10.24 (s, 1H), 8.69 (d, J=7.7 Hz, 1H), 8.20 (s,1H), 8.15 (d, J=8.4 Hz, 1H), 8.09-8.02 (m, 4H), 7.86 (d, J=8.4 Hz, 1H),7.78 (d, J=8.4 Hz, 1H), 7.65 (d, J=8.5 Hz, 2H), 7.57-7.47 (m, 3H),7.40-7.37 (m, 1H), 7.34-7.30 (m, 1H), 6.04 (t, J=5.8 Hz, 1H), 5.48 (brs, 2H), 5.22 (s, 2H), 4.57-4.51 (m, 1H), 4.40-4.33 (m, 2H), 4.25-4.14(m, 4H), 4.03-3.98 (m, 2H), 3.85 (dd, J=10.7, 7.6 Hz, 1H), 3.79 (dd,J=10.3, 8.6 Hz, 1H), 3.66 (t, J=5.2 Hz, 2H), 3.09-2.97 (m, 2H),2.75-2.57 (m, 4H), 2.11-2.06 (m, 1H), 2.01-1.96 (m, 2H), 1.78-1.70 (m,1H), 1.68-1.58 (m, 1H), 1.53-1.40 (m, 2H), 0.95 (d, J=6.8 Hz, 3H), 0.94(d, J=6.8 Hz, 3H). HRMS m/z 924.3672 [(M+H)⁺ calcd for C₄₉H₅₆Cl₂N₇O₇924.3613].

Diisopropylethylamine, DIPEA (10 mg, 0.0774 mmol) in DMF (3 mL) wasadded to 55h (74 mg, 0.0712 mmol) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (33 mg, 0.107 mmol),and the resulting mixture stirred at r.t., under nitrogen. After 5.5 hadditional portions of DIPEA (0.9 mg, 0.00693 mmol) and2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (4.4 mg, 0.0143 mmol)were added. After another 1 h an additional portion of2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (8 mg, 0.0259 mmol)was added and the mixture kept at −20° C. overnight. After 15 h themixture was warmed to r.t. and an additional portion of2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (4.4 mg, 0.0143 mmol)added. After another 1 h an additional portion of2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (7.5 mg, 0.0243 mmol)was added. After 1 h hexanes:EtOAc 95:5 (25 mL) were added followed byDCM (5 mL) and the mixture was stirred for 20 min. Over this period asolid precipitated out of solution. The mixture was left to settle andthe solvents then decanted. Hexanes:DCM 95:5 were then added, thesuspension stirred, left to settle and the solvents decanted and thesolid dried. Purification by preparative HPLC (column: Synergi-MAX RP4μ, 21.20×250 mm; flow rate: 13 mL/min; mobile phase: solvent A: H₂O/TFApH 2.47, solvent B: MeCN/H₂O 90:10; method: isocratic, solvent A:solventB 35:65, 35 min; wavelength: 254 nm, 330 nm) gaveN—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide55 (13.7 mg, 17%, HPLC purity: 92.5%) as a pale yellow powder. ¹H NMR δ(400 MHz, DMSO-d₆) 10.35 (s, 1H), 10.02 (s, 1H), 8.19 (s, 1H), 8.15 (d,J=8.3 Hz, 1H), 8.08 (d, J=8.1 Hz, 2H), 8.02 (s, 1H), 7.86 (s, 1H),7.81-7.77 (m, 2H), 7.66 (d, J=8.5 Hz, 2H), 7.57-7.53 (m, 1H), 7.50-7.47(m, 3H), 7.40-7.36 (m, 1H), 7.33-7.30 (m, 1H), 6.99 (s, 2H), 5.97 (t,J=5.5 Hz, 1H), 5.40 (br s, 2H), 5.21 (s, 2H), 4.42-4.32 (m, 3H),4.22-4.14 (m, 4H), 4.03-3.98 (m, 2H), 3.87-3.77 (m, 2H), 3.34 (t, J=7.0Hz, 2H), 3.07-3.05 (m, 2H), 2.76-2.59 (m, 4H), 2.22-2.08 (m, 2H),2.02-1.92 (m, 3H), 1.74-1.57 (m, 2H), 1.51-1.33 (m, 6H), 1.23-1.14 (m,3H), 0.85 (d, J=6.7 Hz, 3H), 0.82 (d, J=6.8 Hz, 3H). HRMS m/z 1115.4170[(M−H)⁺ calcd for C₅₉H₆₅Cl₂N₈O₁₀ 1115.4206].

Example 6N—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(6-((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)hexanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide56

(S)-tert-Butyl8-(6-((S)-1-(chloromethyl)-5-(4-nitrobenzyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate54g (829 mg, 1.00 mmol) was reduced (Zn/NH₄Cl) to corresponding aniline(by the method reported for the synthesis of 54h above) and dissolved indry DMA (3 mL). To this solution was added a mixture formed by stirring(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid(Fmoc-L-citrulline, 1.19 g, 3.00 mmol) and2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline EEDQ (0.99g, 4.00 mmol)in dry DMA (4 mL) at r.t. for 40 min. The final reaction mixture wasstirred at r.t. and under a nitrogen atmosphere for 19 h. The mixturewas poured into water and stirred at r.t. for 5 h. The solid wascollected, washed with water several times, dried, and purified by asilica column chromatography (DCM-MeOH gradient from 0-5%) to give(S)-tert-butyl8-(6-((S)-5-(4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate56a (0.91 g, 77%) as a pure beige solid, mp 172° C.; ¹H NMR [(CD₃)₂SO] δ10.10 (s, 1H), 8.22-8.11 (m, 2H), 7.94-7.62 (m, 8H), 7.59-7.46 (m, 3H),7.46-7.27 (m, 5H), 7.04 (s, 1H), 6.69 (s, 1H), 6.39 (br s, 1H), 5.99 (t,J=5.6 Hz, 1H), 5.51-5.32 (m, 3H), 5.22 (s, 2H), 4.42-3.90 (m, 10H),3.88-3.75 (m, 1H), 3.79 (s, 3H), 3.53-3.18 (m, 3H), 3.15-2.87 (m, 2H),2.66-2.44 (m, 2H, partially obscured by DMSO peak), 2.11-1.20 (m, 14H),1.31 (s, 9H). Anal. (C₆₅H₇₂ClN₇O₁₂.½H₂O) Calc: C, 65.73; H, 6.20; N,8.26. Found: C, 65.64; H, 6.19; N, 8.27.

To a stirred solution of N-Fmoc 56a (0.91 g, 0.77 mmol) in dry DMA (9mL) at 0° C. under a nitrogen atmosphere was added a solution ofpiperidine in DMA (1.0 mmol per mL solution) (3.85 mL, 3.85 mmol). Afteraddition, the mixture was stirred further at this temperature for 2 hand then poured into a mixture of ethyl acetate-petroleum ether (1:10)(150 mL) and stirred at 0° C. for 30 min. The solvent was decanted fromthe insoluble material and discarded. The wash step was repeated withmore ethyl acetate-petroleum ether (1:3) (2×150 mL) at r.t. The solidwas collected, washed with ethyl acetate-petroleum ether (1:3), anddried to give (S)-tert-butyl8-(6-((S)-5-(4-((S)-2-amino-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate56b as a colorless solid (0.73 g, 99%); mp 223° C. (dec.); ¹H NMR[(CD₃)₂SO] δ 10.60-9.30 (br s, 3H), 8.24-8.12 (m, 2H), 7.84 (d, J=8.2Hz, 1H), 7.70 (d, J=8.5 Hz, 2H), 7.54 (br t, J=7.5 Hz, 1H), 7.50 (d,J=8.5 Hz, 2H), 7.39 (br t, J=7.6 Hz, 1H), 7.04 (s, 1H), 6.69 (s, 1H),6.39 (br s, 1H), 5.93 (t, J=5.7 Hz, 1H), 5.47-5.28 (m, 3H), 5.21 (s,2H), 4.36 (t, J=10.8 Hz, 1H), 4.28-4.13 (m, 2H), 4.10-3.92 (m, 3H),3.90-3.77 (m, 1H), 3.79 (s, 3H), 3.54-3.20 (m, 3H, partially obscured bywater peak), 3.06-2.89 (m, 2H), 2.70-2.49 (m, 3H, partially obscured byDMSO peak), 2.10-1.25 (m, 14H), 1.31 (s, 9H). Anal. (C₅₀H₆₂ClN₇O₁₀.¾H₂O)Calc: C, 61.91; H, 6.60; N, 10.11. Found: C, 62.05; H, 6.96; N, 10.08.

A mixture of amine 56b (0.73 g, 0.763 mmol) and(S)-2,5-dioxopyrrolidin-1-yl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanoate(Fmoc-Val-Osu, 0.50 g, 1.15 mmol) in dry DMA (7 mL) was stirred at r.t.and under a nitrogen atmosphere for 18 h. Ethyl acetate-petroleum ether(1:2) (100 mL) was added and the mixture was stirred at r.t. for 30 min.Solvents were decanted from the insoluble material and the wash step wasrepeated with more ethyl acetate-petroleum ether (1:1) (2×100 mL). Thecolorless solid was dried to give (S)-tert-butyl8-(6-((S)-5-(4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate56c (0.89 g, 91%); mp 191° C. (dec.); ¹H NMR [(CD₃)₂SO] δ 10.11 (s, 1H),8.22-8.08 (m, 3H), 7.92-7.81 (m, 3H), 7.74 (t, J=7.4 Hz, 2H), 7.65 (d,J=8.4 Hz, 2H), 7.54 (br t, J=7.2 Hz, 1H), 7.48 (d, J=8.4 Hz, 2H),7.46-7.35 (m, 4H), 7.32 (br t, J=7.4 Hz, 2H), 7.04 (s, 1H), 6.69 (s,1H), 6.39 (br s, 1H), 5.97 (br s, 1H), 5.47-5.34 (m, 3H), 5.21 (s, 2H),4.53-4.13 (m, 7H), 4.10-3.75 (m, 5H), 3.79 (s, 3H), 3.53-3.20 (m, 3H,partially obscured by water peak), 3.10-2.87 (m, 2H), 2.69-2.45 (m, 2H,partially obscured by DMSO peak), 2.10-1.25 (m, 15H), 1.31 (s, 9H), 0.88(d, J=6.8 Hz, 3H), 0.85 (d, J=6.7 Hz, 3H). Anal. (C₇₀H₈₁ClN₈O₁₃.¾H₂O)Calc: C, 65.10; H, 6.44; N, 8.68. Found: C, 64.85; H, 6.48; N, 8.67.

To a stirred solution of N-Fmoc compound 56c (0.89 g, 0.70 mmol) in dryDMA (6 mL) at 0° C. under a nitrogen atmosphere was added a solution ofpiperidine in DMA (1.0 mmol per mL solution) (3.48 mL, 3.48 mmol). Afteraddition, the mixture was stirred further at this temperature for 1.5 h.A mixture of ethyl acetate-petroleum ether (1:2) (90 mL) was added andthe mixture was stirred at 0° C. for 10 min. The solvent layer wasdecanted from the insoluble material and discarded. The wash step wasrepeated with more ethyl acetate-petroleum ether (1:2) (2×90 mL) at r.t.The colorless solid left behind was dried to give (S)-tert-butyl8-(6-((S)-5-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate56d (0.68 g, 93%); mp 225° C. (dec.); ¹H NMR [(CD₃)₂SO] δ 10.16 (s,exchangeable with D₂O, 1H), 8.23-8.07 (m, 3H, reduced to 2H after D₂O),7.84 (d, J=8.2 Hz, 1H), 7.65 (d, J=8.5 Hz, 2H), 7.59-7.45 (m, 3H), 7.37(br t, J=7.5 Hz, 1H), 7.04 (s, 1H), 6.69 (s, 1H), 6.38 (br s,exchangeable with D₂O, 1H), 5.96 (t, J=5.8 Hz, exchangeable with D₂O,1H), 5.45-5.30 (m, 3H, reduced to 1H as a d after D₂O, J 9.6 Hz), 5.21(s, 2H), 4.41 (br s, became dd after D₂O, J=8.4, 5.4 Hz, 1H), 4.36 (brt, J=10.7 Hz, 1H), 4.26-4.13 (m, 2H), 4.10-3.91 (m, 3H), 3.88-3.76 (m,1H), 3.79 (s, 3H), 3.53-3.43 (m, 1H), 3.41-3.20 (m, 2H), 3.09-2.88 (m,3H), 2.70-2.50 (m, 2H, partially obscured by DMSO peak), 2.10-1.20 (m,15H), 1.31 (s, 9H), 0.88 (d, J=6.9 Hz, 3H), 0.79 (d, J=6.8 Hz, 3H), 2Hnot observed. Anal. (C₅₅H₇₁ClN₈O₁₁.H₂O) Calc: C, 61.53; H, 6.85; N,10.44. Found: C, 61.39; H, 7.11; N, 10.15.

A mixture of amine 56d (0.68 g, 0.64 mmol) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (maleimido-Osu, 0.50g, 1.61 mmol) in dry DMA (6 mL) was stirred at 0° C. under a nitrogenatmosphere for 1 h. A mixture of ethyl acetate-petroleum ether (1:2) (90mL) was added and the mixture was stirred at 0° C. for 15 min. Thesolvent layer was decanted from the insoluble material and discarded.The wash step was repeated with more ethyl acetate-petroleum ether (1:1)(90 mL) and then pure ethyl acetate (50 mL) at r.t. The beige solid leftbehind was dried to give (S)-tert-butyl8-(6-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate52 (0.72 g, 90%); HPLC: 96.7% pure; mp 210° C. (dec.); ¹H NMR [(CD₃)₂SO]δ 10.03 (s, exchangeable with D₂O, 1H), 8.23-8.11 (m, 2H), 8.09 (d,J=7.3 Hz, exchangeable with D₂O, 1H), 7.85 (d, J=8.5 Hz, 1H), 7.80 (d,J=8.3 Hz, exchangeable with D₂O, 1H), 7.66 (d, J=8.6 Hz, 2H), 7.59-7.44(m, 3H), 7.38 (br t, J=7.6 Hz, 1H), 7.04 (s, 1H), 6.99 (s, 2H), 6.69 (s,1H), 6.38 (br s, exchangeable with D₂O, 1H), 5.99 (t, J=5.5 Hz,exchangeable with D₂O, 1H), 5.49-5.34 (m, 3H, reduced to 1H as d afterD₂O, J 9.5 Hz), 5.20 (s, 2H), 4.44-4.30 (m, 2H), 4.26-4.13 (m, 3H),4.10-3.91 (m, 3H), 3.88-3.76 (m, 1H), 3.79 (s, 3H), 3.53-3.44 (m, 1H),3.41-3.20 (m, partially obscured by water peak, 4H), 3.09-2.88 (m, 2H),2.66-2.42 (m, partially obscured by DMSO peak, 2H), 2.25-1.24 (m, 21H),1.31 (s, 9H), 1.24-1.11 (m, 2H), 0.86 (d, J=6.8 Hz, 3H), 0.82 (d, J=6.7Hz, 3H). Anal. (C₅₅H₇₁ClN₈O₁₁.H₂O) Calc: C, 61.53; H, 6.85; N, 10.44.Found: C, 61.39; H, 7.11; N, 10.15.

To a stirred solution of N-^(t)Boc derivative 52 (125 mg, 0.10 mmol) inDCM (10 mL) at −10 to −12° C. (bath temperature) was added dropwise over10 min a solution of 2.5% water in TFA (10 mL). After addition themixture was stirred further at this temperature for 3 h. Cold (−25° C.)ethyl acetate-petroleum ether (1:10) (300 mL) was added, followed byslow addition at −10° C. (bath temperature) of a saturated aqueoussolution of NaHCO₃ to give pH 6-7. The organic layer was removed and thewash step was repeated with more ethyl acetate-petroleum ether (1:1)(300 mL). The solid was collected, washed successively with water andethyl acetate several times, and dried to give the crude product as apale yellow solid (102 mg). This was purified by preparative HPLC[Genentech] to giveN—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(6-((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)hexanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide56 (4.1 mg, 3.6%); HRMS (ESI) m/z calc. for C₆₀H₇₂ClN₉NaO₁: 1152.4932.found: 1152.4906 [MNa⁺]. Calc. for C₆₀H₇₃ClN₉O₁₁: 1130.5113. found:1130.5077 [MH⁺].

Example 7(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57

At room temperature to a solution of (S)-tert-Butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a (2.00g, 5.99 mmol) in DMF (5 mL) was added benzyl bromide (7.13 mL, 59.90mmol), potassium iodide KI (50 mg, 0.30 mmol) and potassium carbonateK₂CO₃ (4.14 g, 30.00 mmol). See FIG. 9. The mixture was stirred for 2 hand then diluted with ethyl acetate. The precipitate was filtered off.The filtrate was redistributed between ethyl acetate and water. Theaqueous phase was extracted with ethyl acetate three times. The combinedorganic extracts were washed with water and brine, dried over anhydrousNa₂SO₄, and filtered through celite. The solvent was removed by rotaryevaporator and the excess benzyl bromide was pumped off. The resultantresidue was purified by column chromatography using a mixture of ethylacetate and petroleum ether (v/v 1:10) as eluent to give (S)-tert-butyl5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate 57aas a white solid (1.97 g, 78%); mp 186-188° C. ¹H NMR (CDCl₃) δ 8.29 (d,J=8.3 Hz, 1H), 7.86 (br s, 1H), 7.65 (d, J=8.29 Hz, 1H), 7.55-7.49 (m,3H), 7.45-7.41 (m, 2H), 7.38-7.31 (m, 2H), 5.26 (s, 2H), 4.26 (br s,1H), 4.13 (t, J=10.8 Hz, 1H), 4.00-3.92 (m, 2H), 3.44 (t, J=10.5 Hz,1H), 1.61 (s, 9H) ppm. LRMS (APCI) found m/z 424.8 (M+H). C₂₅H₂₇ClNO₃requires 424.2. (Boger D., Ishizakilb T., Kitos P. and Suntornwat O.,(1990) J. Org. Chem., 55, 5823-5832.)

Further elution with a mixture of ethyl acetate and petroleum ether (v/v1:1) gave the cyclopropyl product shown in FIG. 9 as a yellow oil (345mg, 19%). (Lajiness J. and Boger D., (2011) J. Org. Chem., 76, 583-587.)

To a solution of 57a (1.60 g, 3.77 mmol) in DCM (15 mL) cooled in an icebath was added 4N HCl in dioxane (40 mL). The mixture was allowed towarm up to room temperature and stirred for 3 h. All volatile componentswere pumped off to give(S)-5-(benzyloxy)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole 57b asa hydrochloride salt. The salt was dissolved in THF (15 mL) and cooleddown in an ice bath. Glutaric anhydride (646 mg, 5.66 mmol), DMAP (46mg, 0.38 mmol) and pyridine (5 mL) were added and the resultant mixturewas stirred for 4 h at room temperature. After all the volatilecomponents were pumped off, the residue was dissolved in dilute aqNaHCO₃ and washed 3 times with ethyl acetate. The aqueous phase wasacidified using 1N HCl to a pH of 2 and extracted with ethyl acetatethree times. The combined ethyl acetate extracts were washed with waterand brine, dried over anhydrous Na₂SO₄, and filtered through a silicagel pad washing with a mixture of MeOH and ethyl acetate (v/v 1:10). Thesolvent was removed to give(S)-5-(5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57c as an off-white solid (978 mg, 59%).

At −10° C., to a solution of 57c (978 mg, 2.23 mmol) in THF (20 mL) wasadded 25% aqueous ammonium formate (20 mL) followed by Pd—C catalyst(10%, wet, 500 mg) and the mixture was stirred at −10° C. for 7 h. MorePd—C catalyst (500 mg) was added and the mixture was stirred at the sametemperature overnight. The catalyst was filtered off through celite andthe celite was washed with THF. The THF was pumped off from the filtrateand the remaining aqueous solution was extracted with ethyl acetatethree times. The combined extracts were washed with water and brine,dried over anhydrous Na₂SO₄ and filtered. Removal of solvent gave(S)-5-(1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 53h as an off-white solid (487 mg, 63%); ¹H NMR (DMSO) δ 12.08 (brs, 1H), 10.35 (br s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.98 (s, 1H), 7.77 (d,J=8.3 Hz, 1H), 7.50-7.46 (m, 1H), 7.33-7.29 (m, 1H), 4.30 (t, J=10.5 Hz,1H), 4.14-4.12 (m, 2H), 3.98 (dd, J=2.8, 10.9 Hz, 1H), 3.78 (dd, J=7.8,10.8 Hz, 1H), 2.63-2.54 (m, 2H), 2.34 (t, J=7.4 Hz, 2H), 1.99-1.83 (m,2H) ppm. LRMS (APCI) found m/z 348.6 (M+H). C₁₈H₁₉ClNO₄ requires 348.1.

To a solution of 53h (500 mg, 1.44 mmol) in THF (15 mL) was addedtetrazole (3% in acetonitrile, 51 mL, 17.25 mmol) followed bydi-tert-butyl-N,N-diisopropyl phosphoramidite (5.73 mL, 17.25 mmol). Themixture was stirred at room temperature overnight then cooled in an icebath and H₂O₂(30% aqueous solution, 3.53 mL, 34.5 mmol) was addeddropwise. The resultant mixture was allowed to warm up to roomtemperature and stirred for 5 h. The reaction was quenched by theaddition of 10% aqueous sodium sulphite with cooling in an ice bath.Organic volatiles were removed by rotary evaporator to give an aqueousphase containing suspended oil. Petroleum ether was added and themixture was stirred for half an hour. The precipitate which formed wascollected by filtration, washed with water and petroleum ether, anddried under vacuum to give(S)-5-(1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57d (660 mg, 85%) as an off-white foam; ¹H NMR (DMSO) δ 12.07 (brs, 1H), 8.56 (s, 1H), 8.04 (d, J=8.2 Hz, 1H), 7.93 (d, J=8.4 Hz, 1H),7.60-7.56 (m, 1H), 7.50-7.46 (m, 1H), 4.38 (t, J=9.8 Hz, 1H), 4.32-4.26(m, 1H), 4.20-4.18 (m, 1H), 4.02 (dd, J=2.9, 11.0 Hz, 1H), 3.90 (dd,J=7.1, 11.0 Hz, 1H), 2.67-2.53 (m, 2H), 2.34 (t, J=7.4 Hz, 2H),1.87-1.78 (m, 2H), 1.481 and 1.476 (2×s, 18H) ppm. ³¹P NMR (DMSO) δ−15.46 ppm. HRMS (ESI) found m/z 562.1719 (M+Na). C₂₆H₃₅ClNNaO₇Prequires 562.1732.

To a suspension of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid(1.00 g, 4.73 mmol) in DCM (20 mL) cooled in an ice bath was added adrop of DMF and then oxalyl chloride (2.03 mL, 23.67 mmol) dropwise. SeeFIG. 10. The mixture was allowed to warm up to room temperature andstirred overnight giving a dark brown solution. All volatile componentswere removed by rotary evaporator and then high vacuum pump. Theresultant residue was dissolved in DCM (5 mL) and the solvent wasremoved by rotary evaporator and then high vacuum pump. The abovedissolving and removal procedure was repeated once more to give crude6-maleimidocaproyl chloride as dark brown oil. To a solution oftert-butyl methyl(2-(methylamino)ethyl)carbamate (891 mg, 4.73 mmol) inDCM (5 mL) cooled in an ice bath was added dropwise a solution of theabove-made 6-maleimidocaproyl chloride in DCM (20 mL). The resultantmixture was allowed to warm up to room temperature and stirredovernight. The DCM was removed and the residue was dissolved in ethylacetate. The solution was washed with aqueous NaHCO₃, cold aqueous 5%citric acid, and brine, then dried over anhydrous Na₂SO₄, and filteredthrough a silica gel pad washing with ethyl acetate. Solvent was removedto give tert-butyl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57e as brown oil (1.33 g, 74%); ¹H NMR (DMSO) (mixture of rotamers) δ7.006 & 7.005 (2×s, 1H), 3.39-3.36 (m, 4H), 3.29-3.25 (m, 2H), 2.92-2.75(m, 6H, 2NMe), 2.21 (t, J=7.38 Hz, 2H), 1.50-1.44 (m, 4H), 1.37 (s, 9H),1.24-1.16 (m, 2H) ppm. HRMS (ESI) found m/z 382.2338 (M+H). C₁₉H₃₂N₃O₅requires 382.2336.

To a solution of 57e (274 mg, 0.72 mmol) in DCM (5 mL) cooled in an icebath was added TFA (5 mL) dropwise. The mixture was stirred at the sametemperature for 2 h before all volatile components were removed byrotary evaporator and then high vacuum pump. The resultant residue,6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methyl-N-(2-(methylamino)ethyl)hexanamidetrifluoroacetate 57f, was used as it was.

At room temperature, 51a (200 mg, 0.60 mmol) was dissolved in DCM (5 mL)and DIPEA (0.3 mL, 1.72 mmol) was added followed by 4-nitrophenylchloroformate (145 mg, 0.72 mmol) to form (S)-tert-butyl1-(chloromethyl)-5-((4-nitrophenoxy)carbonyloxy)-1H-benzo[e]indole-3(2H)-carboxylate57g. After the mixture was stirred for 5 h, a solution of the crude 57fin DCM (5 mL) and DIPEA (0.7 mL, 4.02 mmol) was added to give a brightyellow solution, which was stirred overnight. All volatile componentswere removed. The residue was dissolved in ethyl acetate and washed withaqueous 5% ammonia and brine. The crude material obtained was furtherpurified by column chromatography using a mixture of ethyl acetate, DCM,and petroleum ether (v/v/v 1:2:1), followed by a mixture of ethylacetate and DCM (v/v 1:2) as eluent to give (S)-tert-butyl1-(chloromethyl)-5-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl)(methyl)carbamoyloxy)-1H-benzo[e]indole-3(2H)-carboxylate57h (223 mg, 58%) as an off-white solid; mp 53-56° C. ¹H NMR (CDCl₃)(mixture of rotamers) δ 8.04 (br s, 1H), 7.86-7.78 (m, 1H), 7.72-7.68(m, 1H), 7.53-7.48 (m, 1H), 7.40-7.34 (m, 1H), 6.67 (s, 2H), 4.25 (br s,1H), 4.46-4.10 (m, 1H), 4.06-3.98 (m, 1H), 3.92-3.72 (apparent d, J=11.2Hz, 1H), 3.72-3.42 (m, 7H), 3.28, 3.10, 3.09, 2.99 (4×s, 6H, 2NMe),2.38-2.21 (m, 2H), 1.67-1.54 (m, 4H), 1.57 (s, 9H), 1.33-1.25 (m, 2H)ppm. HRMS (ESI) found m/z 641.2728 (M+H). C₃₃H₄₂ClN₄O₇ requires641.2737.

To a solution of 57h (110 mg, 0.17 mmol) in DCM (2 mL) cooled in an icebath was added TFA (2 mL) dropwise. The mixture was stirred at the sametemperature for 2 h and then all volatile components were removed. Theresultant residue was redistributed between ethyl acetate and colddilute aqueous NaHCO₃. The aqueous phase was extracted with ethylacetate three times. The combined organic extracts were washed withwater followed by brine, dried over anhydrous Na₂SO₄, and filteredthrough celite. The solvent was removed to give(S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57i as a yellow solid (86 mg, 92%), which was used as it was withoutfurther purification; ¹H NMR (CDCl₃) (mixture of rotamers) δ 7.76 (d,J=8.4 Hz, 1H), 7.63-7.59 (m, 1H), 7.48-7.41 (m, 1H), 7.25-7.20 (m, 1H),6.79 (s, 1H), 6.68 (s, 2H), 4.01-3.94 (m, 1H), 3.88-3.78 (m, 3H),3.74-3.68 (m, 2H), 3.62-3.47 (m, 5H), 3.28, 3.10, 3.06, 3.00 (4×s, 6H,2NMe), 2.38-2.21 (m, 2H), 1.69-1.50 (m, 4H), 1.33-1.25 (m, 2H) ppm. HRMS(ESI) found m/z 541.2217 (M+H). C₂₈H₃₄ClN₄O₅ requires 541.2212.

To a solution of 57i (83 mg, 0.15 mmol) in DMA (3 mL) cooled in an icebath was added(S)-5-(1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoic acid 57d (99 mg, 0.18 mmol) followed by EDCIhydrochloride (88 mg, 0.46 mmol) and then p-toluenesulfonic acid (2.6mg, 0.015 mmol). See FIG. 11. The mixture was allowed to warm up to roomtemperature and stirred overnight. The mixture was redistributed betweenethyl acetate and cold dilute aqueous NaHCO₃. The aqueous phase wasextracted with ethyl acetate three times. The combined organic extractswere washed with water followed by brine, dried over anhydrous Na₂SO₄,and filtered through celite. The solvent was removed and the resultantresidue was triturated with petroleum ether. The solid obtained wasre-precipitated from DCM and isopropanol to give(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57j as a yellow solid (116 mg, 71%); mp 81° C. (dec.). ¹H NMR (CDCl₃)(mixture of rotamers) δ 8.63 (s, 1H), 8.36 (s, 1H), 8.23 (d, J=8.5 Hz,1H), 7.89-7.82 (m, 1H), 7.72-7.67 (m, 2H), 7.54-7.50 (m, 2H), 7.43-7.39(m, 2H), 6.66 (s, 2H), 4.34-4.25 (m, 4H), 4.10-4.05 (m, 2H), 3.98-3.93(m, 2H), 3.72-3.69 (m, 2H), 3.50-3.46 (m, 5H), 3.28, 3.10, 3.09, 2.99(4×s, 6H, 2NMe), 2.79-2.73 (m, 2H), 2.70-2.62 (m, 2H), 2.38-2.32 (m,1H), 2.27-2.20 (m, 3H), 1.67-1.54 (m, 3H), 1.56 (s, 9H), 1.55 (s, 9H),1.33-1.25 (m, 4H) ppm. ³¹P NMR (CDCl₃) δ −15.71 ppm. HRMS (ESI) foundm/z 1084.3755 (M+Na). C₅₄H₆₆Cl₂N₅NaO₁₁P requires 1084.3766.

To a solution of 57j (55 mg, 0.052 mmol) in DCM (1 mL) cooled in an icebath was added TFA (1 mL) dropwise. The mixture was stirred at the sametemperature for 1.5 h and then all volatile components were removed. Theresultant residue was re-precipitated from DCM and ethyl acetate to give(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57 as a grey solid (33 mg, 67%, HPLC purity: 89%); mp 191-194° C.(dec.). ¹H NMR (DMSO) (mixture of rotamers) δ 8.49 (s, 1H), 8.23-8.21(m, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.95-7.76 (m, 3H), 7.59-7.53 (m, 2H),7.45-7.39 (m, 2H), 6.97, 6.96, 6.94, 6.90 (4×s, 2H in total, maleimidylgroup), 4.34-4.21 (m, 4H), 4.06-4.01 (m, 2H), 3.95-3.85 (m, 2H),3.71-3.61 (m, 2H), 3.54-3.30 (m, 5H), 3.23, 3.18, 3.04, 3.00, 2.97,2.95, 2.89, 2.85 (8×s, 6H in total, 2NMe), 2.37-2.28 (m, 2H), 2.19 (d,J=7.4 Hz, 1H), 2.00-1.95 (m, 2H), 1.50-1.40 (m, 5H), 1.25-1.15 (m, 5H)ppm. ³¹P NMR (DMSO) δ −5.79 ppm. HRMS (ESI) found m/z 972.2488 (M+Na).C₄₆H₅₀Cl₂N₅NaO₁₁P requires 972.2514.

Example 8(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58

A mixture of (S)-tert-butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a(3.338 g, 10 mmol), 4-methylpiperazine-1-carbonyl chloride hydrochloride(5.98 g, 30 mmol), Et₃N (3.5 g, 35 mmol) and DMAP (1.34 g, 11 mmol) inCH₂Cl₂ (80 mL) was stirred at room temperature for 2 days. See FIG. 12.The mixture was washed with water and the solvent was dried and removedunder vacuum, to give (S)-tert-butyl1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indole-3(2H)-carboxylate58a (Boger D. L. et al, Synthesis, (1999), 1505-1509) in quantitativeyield: mp 98° C.; ¹H NMR (CDCl₃) δ 8.11 (br, 1H), 7.84 (d, J=8.4 Hz,1H), 7.70 (d, J=8.4 Hz, 1H), 7.50 (ddd, J=8.2, 6.9, 1.1 Hz, 1H), 7.37(ddd, J=8.1, 6.9, 1.0 Hz, 1H), 4.34-4.20 (m, 1H), 4.17-4.10 (m, 1H),4.01-3.98 (m, 1H), 3.94 (dd, J=9.6, 2.4 Hz, 1H), 3.87-3.80 (br, 2H),3.68-3.60 (br, 2H), 3.47 (t, J=10.8 Hz, 1H), 2.57-2.48 (m, 4H), 2.83 (s,3H), 1.58 (s, 9H); MS (APCI+) m/z 461.2 MH⁺. Anal. Calcd forC₂₄H₃₀ClN₃O₄: C, 62.7; H, 6.6; N, 9.1. Found: C, 62.5; H, 6.8; N, 9.2%.

A solution of 58a (2.30 g, 5 mmol) in CH₂Cl₂ (50 mL) was treated withexcess trifluoroacetic acid (TFA) at 0° C. for 4 h, and the mixture wasneutralized with cold aq. NH₃. Dilution with hexanes resulted in theprecipitation of a solid which was collected by filtration, washed withwater and hexane, and dried to give(S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58b (1.60 g, 89%): mp 144-147° C.; ¹HNMR (CDCl₃) δ 7.69 (d, J=8.4 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.45 (ddd,J=8.3, 6.9, 1.2 Hz, 1H), 7.25 (ddd, J=8.4, 6.8, 1.2 Hz, 1H), 6.82 (s,1H), 5.30 (s, 1H), 4.17-4.05 (m, 2H), 4.03-3.96 (m, 2H), 3.89-3.77 (m,4H), 3.54 (t, J=10.9 Hz, 1H), 3.20-2.90 (m, 4H), 2.76 (s, 3H). Anal.Calcd for C₁₉H₂₂ClN₃O₂: C, 63.4; H, 6.2; N, 11.7. Found: C, 63.2; H,6.2; N, 11.5%.

A solution 55a (4.689 g, 10 mmol) in dioxane (30 mL) was treated withHCl (4M in dioxane, 10 mL) and the mixture was stirred overnight at roomtemperature. Ammonium hydroxide was added, the solvent was removed togive(S)-1-(chloromethyl)-5-(4-nitrobenzyloxy)-2,3-dihydro-1H-benzo[e]indole54f which was mixed with glutaric anhydride (3.4 g, 30 mmol) in CH₂Cl₂(50 mL). After cooling to 0° C., Et₃N (5.05 g, 50 mmol) was added andthe mixture was allowed to warm slowly and was stirred overnight at roomtemperature. Dilute HCl was added to give a solid which was collected byfiltration, washed with water and CH₂Cl₂, and dried to give(S)-5-(1-(chloromethyl)-5-(4-nitrobenzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 58c (2.95 g, 61%): ¹H NMR (DMSO-d₆) δ 12.07 (br s, 1H), 8.30 (br d,J=8.8 Hz, 2H), 8.24 (d, J=8.1 Hz, 1H), 8.17 (s, 1H), 7.89-7.85 (m, 3H),7.57 (ddd, J=8.2, 6.9, 1.1 Hz, 1H), 7.43 (ddd, J=8.1, 7.1, 1.0 Hz, 1H),5.46 (s, 2H), 4.34 (t, J=9.7 Hz, 1H), 4.25-4.13 (m, 2H), 4.01 (dd,J=11.0, 2.8 Hz, 1H), 3.85 (dd, J=10.9, 7.4 Hz, 1H), 2.66-2.57 (m, 1H),2.55-2.46 (m, 1H), 2.35 (t, J=7.3 Hz, 2H), 1.87-1.79 (m, 2H).

A mixture of 58b (1.33 g, 3.7 mmol) and 58c (1.63 g, 3.38 mmol) in DMA(25 mL) with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDCI.HCl, 3.26 g, 17.0 mmol) was stirred at room temperature overnight.The mixture was diluted with aq. NaHCO₃ and the resulting precipitatewas washed successively with water and methanol, and dried.Chromatography on silica, eluting firstly with EtOAc/MeOH 9:1, and thenEtOAc/MeOH 4:1 gave(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-nitrobenzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58d (0.98 g, 35%): ¹H NMR (CDCl₃) δ8.35 (s, 1H), 8.33-8.27 (m, 3H), 8.17 (s, 1H), 7.85 (d, J=8.3 Hz, 1H),7.73-7.68 (m, 4H), 7.58-7.49 (m, 2H), 7.45-7.38 (m, 2H), 5.33 (q, J=13.0Hz, 2H), 4.39-4.27 (m, 4H), 4.14-4.06 (m, 2H), 4.01-3.95 (m, 2H),3.83-3.77 (m, 2H), 3.64-3.59 (m, 2H), 3.48 (dt, J=10.5, 7.3 Hz, 2H),2.85-2.65 (m, 4H), 2.55-2.47 (m, 4H), 2.38 (s, 3H), 2.28-2.21 (m, 2H).

A suspension of 58d (0.41 g, 5 mmol) in a mixture of THF (35 mL), MeOH(15 mL), and water (5 mL) at 0° C. was treated with aluminum amalgam (2g) and the stirred mixture was allowed to warm to room temperature over3 h. After dilution with MeOH the mixture was filtered through celiteand the filtrate was evaporated to dryness to give(S)-3-(5-((S)-5-(4-aminobenzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58e (0.31 g, 78%) which was useddirectly in the next step: ¹H NMR (CDCl₃) δ 8.37 (s, 1H), 8.26 (d, J=8.2Hz, 1H), 8.18 (s, 1H), 7.86 (d, J=8.3 Hz, 1H), 7.67 (d, J=8.3 Hz, 1H),7.60 (d, J=8.3 Hz, 1H), 7.49-7.43 (m, 2H), 7.39 (br t, J=7.6 Hz, 1H),7.34-7.26 (m, 3H), 6.68 (d, J=8.3 Hz, 2H), 5.10 (q, J=10.9 Hz, 2H),4.32-4.15 (m, 4H), 4.07-3.97 (m, 2H), 3.94-3.90 (m, 2H), 3.86-3.79 (m,2H), 3.66-3.60 (m, 2H), 3.50-3.40 (m, 2H), 2.77-2.58 (m, 4H), 2.56-2.48(m, 4H), 2.27 (s, 3H), 2.22-2.14 (m, 2H).

A mixture of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 0.58g, 2.3 mmol) and(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid(Fmoc-L-citrulline, 0.69 g, 1.7 mmol) was stirred in dry DMA (10 mL)under nitrogen for 10 min until all solid was dissolved. See FIG. 13.Crude 58e (0.31 g, 0.39 mmol) was added and the stirring was continuedovernight. The mixture was diluted with EtOAc and water was added toprecipitate the product, which was collected by filtration, andtriturated with boiling MeOH to give crude(S)-3-(5-((S)-5-(4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58f (0.62 g, >100%), which was treatedwith piperidine (50 mg, 0.59 mmol) in dry DMF (20 mL) at roomtemperature for 30 min. Dilution with EtOAc, hexanes and water gave aprecipitate which was collected by filtration and washed with hexanesand water, to give crude(S)-3-(5-((S)-5-(4-((S)-2-amino-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58g (0.33 g, 89%, 85% purity by HPLC)which was treated with 1.5 equivalents of Fmoc-Va-OSu (N-α-Fmoc-L-valineN-hydroxysuccinimide ester, 0.23 g, 0.53 mmol) in dry DMA (10 mL)overnight. Dilution with EtOAc and water gave a solid which wascollected by filtration and dried, to give crude(S)-3-(5-((S)-5-(4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58h (0.38 g, 85%): ¹H NMR (DMSO-d₆) δ10.11 (s, 1H), 8.24-8.11 (m, 3H), 7.96 (d, J=8.4 Hz, 1H), 7.90-7.80 (m,4H), 7.74 (t, J=7.4 Hz, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.61-7.53 (m, 2H),7.50-7.36 (m, 6H), 7.32 (t, J=7.5 Hz, 2H), 5.97 (t, J=5.4 Hz, 1H), 5.40(s, 2H), 5.20 (s, 2H), 4.44-4.19 (m, 9H), 4.08-3.81 (m, 5H), 3.74-3.70(br, 2H), 3.50-3.42 (br, 2H), 3.07-2.89 (m, 2H), 2.77-2.55 (m, 4H),2.52-2.36 (m, 4H), 2.25 (s, 3H), 2.03-1.94 (m, 3H), 1.76-1.53 (m, 2H),1.49-1.30 (m, 2H), 0.87 (dd, J=11.2, 6.8 Hz, 6H).

Crude 58h (0.38 g, 0.3 mmol) was reacted with piperidine in DMA at roomtemperature for 1 h and the mixture was diluted with EtOAc and water, togive a solid which was collected and dried to give(S)-3-(5-((S)-5-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58i (0.23 g, 73%, 83% purity by HPLC):¹H NMR (CDCl₃) δ 9.34 (br, 1H), 8.35 (br s, 1H), 8.28 (d, J=8.1 Hz, 1H),8.14 (s, 1H), 8.02 (s, 1H), 7.86 (d, J=8.2 Hz, 1H), 7.75 (d, J=7.5 Hz,1H), 7.70 (d, J=7.6 Hz, 1H), 7.65-7.60 (m, 2H), 7.54-7.47 (m, 2H),7.46-7.33 (m, 4H), 7.29 (td, J=7.4, 1.3 Hz, 1H), 5.20 (br s, 2H),5.17-5.09 (m, 1H), 4.82-4.73 (m, 1H), 4.59-4.50 (m, 1H), 4.33-4.15 (m.3H), 4.07-4.01 (m, 1H), 3.98-3.90 (m, 2H), 3.86-3.76 (m, 3H), 3.65-3.57(m, 2H), 3.55-3.39 (m, 3H), 3.25 (br s, 1H), 2.81-2.60 (m, 3H),2.59-2.41 (m, 6H), 2.37 (s, 3H), 2.27-2.16 (m, 3H), 1.55-1.48 (m, 2H),0.98 (d, J=6.7 Hz, 3H), 0.83 (d, J=6.6 Hz, 3H).

A mixture of crude 58i (0.105 g, 0.1 mmol) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (62 mg, 0.2 mmol) indry DMF (10 mL) was stirred overnight at room temperature and themixture was diluted with EtOAc and water, to give a solid which wascollected and dried to give crude material (70 mg, 71% purity by HPLC)which was purified by prep-HPLC to give(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58. MS m/z 1243.4 [(M+H)⁺ calcd forC₆₅H₇₆Cl₂N₁₀O₁₁ 1243.5].

Example 9(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 59

To a solution of (S)-tert-butyl5-(4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate55e (1.00 g, 1.09 mmol) in DMF (10 mL) was added piperidine (1.08 mL,10.90 mmol). See FIG. 14. The mixture was stirred at room temperaturefor 2 h and then all the volatile components were pumped off. Theresultant residue was triturated with ether to give (S)-tert-butyl5-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate59a as a white solid (700 mg, 92%). ¹H NMR (DMSO) δ 10.17 (s, 1H), 8.17(d, J=8.0 Hz, 1H), 8.12 (d, J=8.4 Hz, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.66(d, J=8.6 Hz, 2H), 7.55-7.49 (m, 3H), 7.37-7.33 (m, 1H), 5.98 (t, J=5.7Hz, 1H), 5.41 (s, 2H), 5.22 (s, 2H), 4.51-4.48 (m, 1H), 4.19-3.98 (m,4H), 3.84-3.80 (m, 1H), 3.08-2.90 (m, 3H), 1.99-1.91 (m, 2H), 1.75-1.65(m, 2H), 1.55 (s, 9H), 1.49-1.33 (m, 2H), 0.89 (d, J=6.8 Hz, 3H), 0.80(d, J=6.8 Hz, 3H) ppm.

A mixture of 59a (688 mg, 0.99 mmol), 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (SuOMC, 320 mg, 1.04mmol), and DIPEA (190 μL, 1.09 mmol) in DMSO (10 mL) was stirred at roomtemperature overnight. All the volatile components were pumped off. Theresultant residue was triturated with ethyl acetate to give(S)-tert-butyl1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indole-3(2H)-carboxylate59b as an off-white solid (832 mg, 95%). ¹H NMR (DMSO) δ 10.03 (s, 1H),8.13-8.08 (m, 2H), 7.83-7.79 (m, 2H), 7.66 (d, J=8.4 Hz, 2H), 7.55-7.48(m, 3H), 7.35 (t, J=7.5 Hz, 1H), 6.99 (s, 2H), 5.97 (t, J=5.4 Hz, 1H),5.41 (s, 2H), 5.21 (s, 2H), 4.43-4.38 (m, 1H), 4.22-3.98 (m, 4H),3.84-3.80 (m, 1H), 3.38-3.33 (m, 3H), 3.08-2.90 (m, 2H), 2.24-2.06 (m,2H), 2.01-1.91 (m, 1H), 1.74-1.14 (m, 10H), 1.55 (s, 9H), 0.86 (d, J=6.8Hz, 3H), 0.83 (d, J=6.7 Hz, 3H) ppm. HRMS (ESI) found m/z 910.3897(M+Na). C₄₆H₅₈ClN₇NaO₉ requires 910.3877.

To a suspension of 59b (100 mg, 0.11 mmol) in DCM (2 mL) cooled in anice bath was added TFA (2 mL). The mixture was stirred in the ice bathfor 3 h. All the volatile components were pumped off. The resultantresidue was dissolved in THF and redistributed between ethyl acetate andcold 5% aqueous ammonia. The aqueous phase was extracted with ethylacetate three times. The combined organic extracts were washed withwater and brine, dried over anhydrous Na₂SO₄, filtered through celiteand the solvent was removed to giveN—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide59c as a greenish-brown solid (80 mg, 90%), which was used directly. ¹HNMR (DMSO) δ 10.02 (s, 1H), 8.09-8.07 (m, 1H), 7.99 (d, J=8.3 Hz, 1H),7.80 (d, J=8.5 Hz, 1H), 7.65 (d, J=8.3 Hz, 2H), 7.56 (d, J=8.3 Hz, 1H),7.45 (d, J=8.4 Hz, 2H), 7.38 (t, J=7.1 Hz, 1H), 7.09 (t, J=7.4 Hz, 1H),6.99 (s, 2H), 5.97 (br s, 1H), 5.41 (s, 2H), 5.16 (s, 2H), 4.46-4.35 (m,1H), 4.21-4.17 (m, 1H), 3.96-3.87 (m, 1H), 3.84-3.80 (m, 1H), 3.70-3.65(m, 1H), 3.60-3.49 (m, 1H), 3.38-3.33 (m, 3H), 3.06-2.92 (m, 2H),2.22-2.08 (m, 2H), 2.02-1.92 (m, 1H), 1.75-1.14 (m, 10H), 0.86 (d, J=6.7Hz, 3H), 0.82 (d, J=6.7 Hz, 3H) ppm. HRMS (ESI) found m/z 810.3332(M+Na). C₄₁H₅₀ClN₇NaO₇ requires 810.3352.

At room temperature, to a solution of 59c (75 mg, 0.095 mmol) and(S)-5-(1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57d (62 mg, 0.11 mmol) in DMA (3 mL) was added EDCI hydrochloride(40 mg, 0.21 mmol) and then para-toluenesulfonic acid (1.6 mg, 0.0095mmol). After the mixture was stirred for 5 h, more EDCI hydrochloride(35 mg, 0.18 mmol) was added and the mixture was stirred overnight. Allthe volatile components were pumped off. The resultant residue wasdissolved in THF and redistributed between ethyl acetate and cold diluteaqueous NaHCO₃. The aqueous phase was extracted with ethyl acetate threetimes. The combined organic extracts were washed with water and brine,dried over anhydrous Na₂SO₄, filtered through celite and the solvent wasremoved to give di-tert-butyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylphosphate 59d as a brown solid (104 mg, 83%), which was used directly.¹H NMR (DMSO) δ 10.03 (s, 1H), 8.60 (s, 1H), 8.19-7.38 (m, 14H), 6.87(s, 2H), 5.97 (br s, 1H), 5.40 (s, 2H), 5.20 (br s, 2H), 4.45-3.82 (m,10H), 3.38-3.33 (m, 3H), 3.07-2.90 (m, 2H), 2.75-2.50 (m, 2H), 2.20-2.08(m, 4H), 2.02-1.92 (m, 2H), 1.75-1.10 (m, 12H), 1.48 (s, 18H), 0.86-0.82(m, 6H) ppm. ³¹P NMR (DMSO) δ −15.46 ppm. HRMS (ESI) found m/z 1331.5071(M+Na). C₆₇H₈₃Cl₂N₈NaO₁₃P requires 1331.5086.

To a suspension of 59d (95 mg, 0.073 mmol) in DCM (2 mL) cooled in anice bath was added TFA (1 mL). The mixture was stirred in the ice bathfor 1.5 h. All the volatile components were pumped off. The resultantresidue was triturated with ethyl acetate to give a bluish grey solid(77 mg, 90%), which was further purified by preparative HPLC (column:Synergi-Max RP 4μ, 250×21.20 mm; mobile phase: A/B=30:70 (A: H₂O-TFA pH2.56, B: 90% acetonitrile in water); flow rate 13 mL/min) to give(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 59 as an off-white solid (5 mg, HPLC purity 97%).¹H NMR (DMSO) δ 10.04 (s, 1H), 8.49 (s, 1H), 8.19-8.09 (m, 4H),7.87-7.81 (m, 2H), 7.66 (d, J=8.5 Hz, 2H), 7.56-7.48 (m, 4H), 7.40-7.36(m, 2H), 6.98 (s, 2H), 6.01 (br s, 1H), 5.43 (br s, 2H), 5.22 (s, 2H),4.42-3.34 (m, 2H), 4.25-4.12 (m, 4H), 4.03-3.98 (m, 2H), 3.88-3.81 (m,2H), 3.38-3.33 (m, 3H), 3.10-2.90 (m, 2H), 2.75-2.55 (m, 4H), 2.20-2.08(m, 2H), 2.00-1.92 (m, 2H), 1.75-1.10 (m, 12H), 0.88-0.81 (m, 6H) ppm.³¹P NMR (DMSO) δ −5.60 ppm. HRMS (ESI) found m/z 1219.3794 (M+Na).C₅₉H₆₇Cl₂N₈NaO₁₃P requires 1219.3834

Example 10N—((S)-1-((S)-1-(4-(((S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy)methyl)phenylamino)-1-oxo-5-ureidopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide60

(S)-1-(Chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 59 was enzymatically dephosphorylated to give 60.

Example 11 2-(pyridin-2-yldisulfanyl)ethyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate61

A solution of triphosgene (136 mg, 0.458 mmol) in dry DCM (10 mL) wasadded to a mixture of (S)-tert-butyl5-amino-1-(chloromethyl)-1H-benzo[e]indole-3 (2H)-carboxylate 61a (150mg, 0.451 mmol) and DMAP (386 mg, 3.16 mmol) in dry DCM (30 mL) at r.t.See FIG. 15. After 45 min a solution of2-(pyridin-2-yldisulfanyl)ethanol (Chem. Eur. J. (2006) 12:3655-3671)(350 mg, 1.87 mmol) in dry DCM (10 mL) was added and the reactionmixture was stirred overnight. After 20 h the mixture was diluted withMeOH (30 mL) and the solvents removed under vacuum. Purification bycolumn chromatography on silica gel using hexanes:DCM 100:0 to 0:100,then DCM:EtOAc 100:0 to 95:5 gave a mixture of compounds 61b andstarting material 2-(pyridin-2-yldisulfanyl)ethanol (389 mg). Thismixture was used in the next step. A solution of TBDMSCl (262 mg, 1.74mmol) in DMF (1.5 mL) was added to a stirred mixture of product 61b,2-(pyridin-2-yldisulfanyl)ethanol, and imidazole (118 mg, 1.74 mmol) inDMF (4 mL) at 0° C. The mixture was warmed to r.t., stirred for 45 minand then diluted with EtOAc and H₂O. The layers were separated and theorganic layer was washed with H₂O (3×), dried (Na₂SO₄) and solventremoved under vacuum. Purification by column chromatography on silicagel using hexanes:DCM 50:50 to 0:100, then DCM:EtOAc 98:2 to 94:6 gave(S)-tert-butyl1-(chloromethyl)-5-((2-(pyridin-2-yldisulfanyl)ethoxy)carbonylamino)-1H-benzo[e]indole-3(2H)-carboxylate61b (190 mg, 77% over two steps from 61a) as a pale yellow foamy solid.¹H NMR δ (400 MHz, CDCl₃) 8.49 (br s, 1H), 8.48-8.47 (m, 1H), 7.84 (d,J=8.4 Hz, 1H), 7.71 (t, J=7.0 Hz, 2H), 7.63 (t, J=7.3 Hz, 1H), 7.54-7.50(m, 1H), 7.40 (ddd, J=8.2, 6.8, 1.1 Hz, 1H), 7.09 (ddd, J=7.3, 4.9, 1.0Hz, 1H), 6.93 (br s, 1H), 4.48 (t, J=6.3 Hz, 2H), 4.31-4.27 (m, 1H),4.15-4.10 (m, 1H), 4.04-3.98 (m, 1H), 3.91 (dd, J=11.1, 2.4 Hz, 1H),3.45 (t, J=10.7 Hz, 1H), 3.13 (t, J=6.3 Hz, 2H), 1.60 (s, 9H).

TFA (4.8 mL) was added slowly to a solution of 61b (180 mg, 0.330 mmol)in DCM (9.5 mL) at 0° C. and the mixture stirred at this temperature for1 h. The reaction mixture was then diluted with DCM and H₂O andneutralized with saturated aqueous NaHCO₃ until pH 7-8. The layers wereseparated and the organic layer washed with H₂O (1×), dried (Na₂SO₄) andsolvent removed under vacuum to give (S)-2-(pyridin-2-yldisulfanyl)ethyl1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate 61c (113 mg,77%) as a yellow solid which was used in the next step withoutpurification. ¹H NMR δ (400 MHz, DMSO-d₆) 9.52 (s, 1H), 8.48-8.46 (m,1H), 7.88 (d, J=8.4 Hz, 1H), 7.83-7.78 (m, 2H), 7.64 (d, J=8.2 Hz, 1H),7.39 (ddd, J=8.1, 6.9, 1.0 Hz, 1H), 7.25 (ddd, J=6.5, 4.8, 2.2 Hz, 1H),7.14 (ddd, J=8.2, 6.8, 1.0 Hz, 1H), 7.09 (s, 1H), 5.94 (br s, 1H), 4.33(t, J=6.2 Hz, 2H), 4.02-3.96 (m, 1H), 3.85 (dd, J=10.8, 3.5 Hz, 1H),3.70 (t, J=9.3 Hz, 1H), 3.63-3.55 (m, 2H), 3.18 (t, J=6.1 Hz, 2H).

A solution of(S)-5-(1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57d (135 mg, 0.250 mmol) in DMA (6 mL) was added to a mixture of61c (110 mg, 0.247 mmol) and EDCI.HCl (116 mg, 0.605 mmol) at r.t.,under nitrogen and mixture stirred overnight. After 18.5 h an additionalsolution of 57d (40 mg, 0.0741 mmol) in DMA (0.5 mL) and solid EDCI.HCl(28.4 mg, 0.148 mmol) were added and the mixture stirred at r.t, undernitrogen. After another 6 h an additional portion of 57d (7 mg, 0.0130mmol) in DMA (0.5 mL) and solid EDCI.HCl (24 mg, 0.125 mmol) were addedand the mixture stirred for a further 2 days and 16.5 h. The mixture wasthen diluted with H₂O and a solid precipitated out. The solid wascollected by filtration, washed with H₂O, saturated aqueous NaHCO₃, H₂Oand hexanes. The solid was dissolved in EtOAc, the solution was washedwith saturated aqueous NaHCO₃ (3×) and H₂O (1×) and then dried (Na₂SO₄)and solvent removed under vacuum. The solid was then re-dissolved in DCMand washed with more saturated aqueous NaHCO₃ (3×), dried (Na₂SO₄) andsolvent removed under vacuum. The solid was then triturated withhexanes:EtOAc 95:5 to 90:10 to give 2-(pyridin-2-yldisulfanyl)ethyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate61d (117 mg, HPLC purity: 86.1%) as a beige solid, which was used in thenext step without further purification. HRMS m/z 989.2289 [(M+Na)⁺ calcdfor C₄₇H₅₃Cl₂N₄NaO₈PS₂ 989.2312].

TFA (1 mL) was added dropwise to a stirred solution of 61d in DCM (2 mL)at 0° C. and the mixture stirred at this temperature for 70 min. Thesolvents were then removed under vacuum at 25° C. The resulting blackresidue was dissolved in DCM and the solution diluted with EtOAc. TheDCM was removed under vacuum to give a suspension in EtOAc. The solidwas collected by filtration, triturated with EtOAc and hexanes and driedto give a green solid. This was further purified by preparative HPLC(column: Synergi-MAX RP 4μ, 21.20×250 mm; flow rate: 12 mL/min; mobilephase: solvent A: H₂O/TFA pH 2.6, solvent B: MeCN/H₂O 90:10; method:gradient, solvent A:solvent B 60:40 to 2:98, 35 min; wavelength: 254 nm,330 nm) to give 61, 16 mg, 8% over two steps from 61c, HPLC purity:97.2%). ¹H NMR δ (400 MHz, DMSO-d₆) 9.69 (s, 1H), 8.57-8.46 (m, 3H),8.09 (d, J=8.3 Hz, 1H), 8.01 (d, J=8.5 Hz, 1H), 7.91 (dd, J=8.3, 2.4 Hz,2H), 7.82 (d, J=3.3 Hz, 2H), 7.59-7.52 (m, 2H), 7.47-7.40 (m, 2H),7.27-7.23 (m, 1H), 4.42-4.22 (m, 8H), 4.06-4.01 (m, 2H), 3.90 (td,J=11.2, 7.4 Hz, 2H), 3.19 (t, J=6.0 Hz, 2H), 2.77-2.59 (m, 4H),2.01-1.94 (m, 2H). 2 protons not observed. ³¹P NMR δ (400 MHz, DMSO-d₆)−6.01. HRMS m/z 877.1053 [(M+Na)⁺ calcd for C₃₉H₃₇Cl₂N₄NaO₈PS₂877.1060]. [α]_(D) ²⁴=−42.3° (c=0.213, DMSO).

Example 12 2-(pyridin-2-yldisulfanyl)propyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate62

Triethylamine, Et₃N (0.92 mL, 6.62 mmol) and triflic anhydride (1.03 mL,6.11 mmol) were added to a stirred solution of 51a (1.70 g, 5.09 mmol)in DCM (160 mL) at 0° C. See FIG. 16. The reaction was stirred at 0° C.for 20 min, then diluted with H₂O, layers separated and the aqueouslayer extracted with DCM (1×). The combined organic layers were dried(Na₂SO₄) and solvent removed under vacuum. Purification by columnchromatography on silica gel using hexanes:EtOAc 100:0 to 95:5 gave(S)-tert-butyl1-(chloromethyl)-5-(trifluoromethylsulfonyloxy)-1H-benzo[e]indole-3(2H)-carboxylate62a (2.20 g, 93%) as a beige foamy solid. ¹H NMR δ (400 MHz, CDCl₃) 8.30(br s, 1H), 8.03 (d, J=8.5 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.62-7.59(m, 1H), 7.53-7.49 (m, 1H), 4.32 (br s, 1H), 4.21-4.15 (m, 1H),4.09-4.03 (m, 1H), 3.92 (dd, J=11.2, 2.8 Hz, 1H), 3.54-3.49 (m, 1H),1.61 (s, 9H).

A solution of 62a (2.15 g, 4.61 mmol) in dry THF (60 mL) was added to amixture of Cs₂CO₃ (2.10 g, 6.44 mmol), BINAP (430 mg, 0.690 mmol) andPd(OAc)₂ (155 mg, 0.690 mmol) in a sealed tube, under nitrogen.Diphenylmethanimine (1.0 mL, 5.98 mmol) was then added to the reactionmixture and nitrogen bubbled through the mixture for 10 min. The sealedtube was heated at 60-65° C. for 4 days. The reaction mixture was thencooled to r.t., diluted with DCM, filtered through celite, the celiteplug washed with DCM until there was no more color in the washings andthe filtrate evaporated under vacuum. Purification of the residue bycolumn chromatography on silica gel using hexanes:DCM 100:0 to 50:50gave (S)-tert-butyl1-(chloromethyl)-5-(diphenylmethyleneamino)-1H-benzo[e]indole-3(2H)-carboxylate 53f (2.09 g, 91%) as a yellow, foamy solid. ¹H NMR δ(400 MHz, DMSO-d₆) 7.85 (d, J=8.4 Hz, 1H), 7.80 (d, J=8.4 Hz, 1H), 7.75(d, J=7.3 Hz, 2H), 7.61-7.57 (m, 1H), 7.54-7.49 (m, 3H), 7.37-7.33 (m,1H), 7.30-7.23 (m, 3H), 7.06 (d, J=6.7 Hz, 2H), 4.14-4.02 (m, 2H),3.99-3.94 (m, 2H), 3.77 (dd, J=10.8, 7.4 Hz, 1H), 1.46 (s, 9H), 1H notobserved. HRMS m/z 497.1984 [(M+H)⁺ calcd for C₃₁H₃₀ClN₂O₂ 497.1990].[α]_(D) ²⁸=−101.5° (c=0.995, DCM).

Glacial acetic acid, HOAc (65 mL) was added to a stirred solution of 53f(1.30 g, 2.62 mmol) in THF and H₂O (195 mL/98 mL) at r.t. and themixture stirred overnight. After 18 h the reaction mixture wasconcentrated under vacuum to remove most of the THF, without heatingabove 30° C. The mixture was then diluted with EtOAc (200 mL), theorganic layer was separated, washed with saturated aqueous NaHCO₃ (4×,until washings were pH 8), dried (Na₂SO₄) and the solvent was removedunder vacuum. Purification of the residue by column chromatography onsilica gel using hexanes:EtOAc 100:0 to 90:10 gave (S)-tert-butyl5-amino-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate 61a (503mg, 58%). ¹H NMR δ (400 MHz, DMSO-d₆) 8.01 (d, J=8.4 Hz, 1H), 7.64 (d,J=8.0 Hz, 1H), 7.40 (ddd, J=8.1, 6.8, 0.9 Hz, 1H), 7.36 (br s, 1H), 7.20(ddd, J=8.1, 6.8, 1.1 Hz, 1H), 5.91 (s, 2H), 4.11-3.91 (m, 4H), 3.66(dd, J=10.6, 8.2 Hz, 1H), 1.53 (s, 9H).

A solution of 2-mercaptopropanoic acid (3.02 g, 28.5 mmol) in dry THF(10 mL) was added dropwise to a stirred suspension of LiAlH₄ (1.29 g,34.0 mmol) in dry THF (40 mL) at 0° C. The reaction mixture was warmedto r.t. and stirred for 3 h. The mixture was then cooled to 0° C. andquenched with H₂O (5 mL) and 5% aqueous NaOH solution (3 mL). Themixture was stirred at 0° C. for 20 min, filtered through celite, thecelite plug washed with Et₂O (3×), the combined organics dried (Na₂SO₄),filtered and solvent removed to give 2-mercaptopropan-1-ol (944 mg)which was used in the next step without purification. A solution of1,2-di(pyridin-2-yl)disulfane (Bioorg. Med. Chem. Lett. (2011)21:4985-4988.) (470 mg, 5.10 mmol) in MeOH (7 mL) was added to asolution of 2-mercaptopropan-1-ol in MeOH (4 mL) at r.t. and the mixturestirred overnight. After 17.5 h the solvent was removed under vacuum.Purification by column chromatography on alumina (neutral) usinghexanes:DCM 50:50 to 0:100, then DCM:EtOAc 99:1 to 75:25 gave2-(pyridin-2-yldisulfanyl)propan-1-ol (528 mg, 18% over two steps from2-mercaptopropanoic acid) as a yellow oil. ¹H NMR δ (400 MHz, CDCl₃)8.50 (ddd, J=5.0, 1.8, 0.9 Hz, 1H), 7.57 (ddd, J=8.0, 7.4, 1.8 Hz, 1H),7.39 (td, J=8.1, 1.0 Hz, 1H), 7.15 (ddd, J=7.4, 5.0, 1.1 Hz, 1H), 5.93(dd, J=8.8, 5.8 Hz, 1H), 3.68 (ddd, J=12.5, 8.8, 3.8 Hz, 1H), 3.40 (ddd,J=12.4, 7.8, 5.8 Hz, 1H), 3.14-3.06 (m, 1H), 1.31 (d, J=6.9 Hz, 3H).

A solution of triphosgene (127 mg, 0.428 mmol) in dry DCM (10 mL) wasadded slowly to a mixture of 61a (250 mg, 0.751 mmol) and DMAP (551 mg,4.51 mmol) in dry DCM (40 mL) at r.t. A yellow solid precipitatedimmediately. After 30 min a solution of2-(pyridin-2-yldisulfanyl)propan-1-ol (420 mg, 2.09 mmol) in dry DCM (6mL) was added and the precipitate dissolved. The reaction mixture wasleft stirring overnight. After 18 h 1M NaOH (30 mL) was added and themixture stirred. The layers were then separated and the organic layerdried (Na₂SO₄), diluted with MeOH (15 mL) and absorbed onto silica gel.The product was eluted using hexanes:DCM 100:0 to 50:50 to 0:100, thenDCM:EtOAc 99:1 to 92:8. The material was then chromatographed again onsilica gel using hexanes:DCM 100:0 to 50:50 to 0:100, then DCM:EtOAc98:2 to 95:5. This gave a mixture of 62b and2-(pyridin-2-yldisulfanyl)propan-1-ol (385 mg). This mixture was used inthe next step. A solution of TBDMSCl (81 mg, 0.535 mmol) in DMF (1 mL)was added to a stirred mixture of 62b and2-(pyridin-2-yldisulfanyl)propan-1-ol, and imidazole (36 mg, 0.535 mmol)in DMF (2 mL) at 0° C. The mixture was warmed to r.t., stirred for 50min and then diluted with EtOAc and H₂O. The mixture was well stirred,the layers separated and the organic layer washed with H₂O (3×), dried(Na₂SO₄) and solvent removed under vacuum. Purification by columnchromatography on silica gel hexanes:DCM 50:50 to 0:100, then DCM:EtOAc95:5 gave (1S)-tert-butyl1-(chloromethyl)-5-((2-(pyridin-2-yldisulfanyl)propoxy)carbonylamino)-1H-benzo[e]indole-3(2H)-carboxylate62b (295 mg, 70% over two steps from compound 61a) as a pale yellowfoamy solid. ¹H NMR δ (400 MHz, CDCl₃) 8.49 (br s, 1H), 8.46 (ddd,J=4.8, 1.7, 0.8 Hz, 1H), 7.86 (d, J=8.4 Hz, 1H), 7.75-7.73 (m, 2H),7.64-7.60 (m, 1H), 7.54 (ddd, J=8.1, 6.9, 1.0 Hz, 1H), 7.42 (ddd, J=8.2,6.8, 1.1 Hz, 1H), 7.08 (ddd, J=7.4, 4.9, 0.8 Hz, 1H), 6.97 (br s, 1H),4.37-4.28 (m, 3H), 4.17-4.11 (m, 1H), 4.05-3.99 (m, 1H), 3.92 (dd,J=11.1, 2.5 Hz, 1H), 3.47 (t, J=10.7 Hz, 1H), 3.38-3.30 (m, 1H), 1.62(s, 9H), 1.41 (d, J=6.9 Hz, 3H). HRMS m/z 582.1265 [(M+Na)⁺ calcd forC₂₇H₃₀ClN₃NaO₄S₂ 582.1258]

TFA (7 mL) was added slowly to a solution of 62b (285 mg, 509 mmol) inDCM (14 mL) at 0° C. and the mixture stirred at this temperature for 1h. The reaction mixture was then diluted with DCM and H₂O andneutralized with saturated aqueous NaHCO₃ until pH 7. The layers wereseparated and the organic layer washed with H₂O (1×), dried (Na₂SO₄) andsolvent removed under vacuum to give 2-(pyridin-2-yldisulfanyl)propyl(S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate 62c (220mg, 94%) as a yellow solid which was used in the next step withoutpurification. ¹H NMR δ (400 MHz, DMSO-d₆) 9.52 (s, 1H), 8.45 (ddd,J=4.8, 1.7, 0.8 Hz, 1H), 7.88 (d, J=8.5 Hz, 1H), 7.85-7.83 (m, 1H),7.79-7.75 (m, 1H), 7.64 (d, J=8.2 Hz, 1H), 7.39 (ddd, J=8.1, 6.8, 1.0Hz, 1H), 7.23 (ddd, J=7.3, 4.8, 1.0 Hz, 1H), 7.15 (ddd, J=8.1, 6.8, 1.0Hz, 1H), 7.08 (s, 1H), 5.93 (br s, 1H), 4.24-4.13 (m, 2H), 4.02-3.96 (m,1H), 3.85 (dd, J=10.8, 3.5 Hz, 1H), 3.70 (t, J=9.3 Hz, 1H), 3.63-3.55(m, 2H), 3.43-3.36 (m, 1H), 1.34 (d, J=6.9 Hz, 3H).

A solution of(S)-5-(1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoicacid 57d (365 mg, 0.676 mmol) in DMA (10 mL) was added to a mixture of62c (215 mg, 0.467 mmol) and EDCI.HCl (268 mg, 1.40 mmol) at r.t. TsOHwas added (16 mg, 0.0929 mmol) and the mixture stirred overnight, undernitrogen (and over 3A molecular sieves). After 27.5 h the mixture wasdiluted with EtOAc and H₂O, well shaken and the layers separated. Theorganic layer was washed with saturated aqueous NaHCO₃ (3×), brine (1×),dried (Na₂SO₄) and solvent removed under vacuum. The resulting residuewas dissolved in DCM and diluted with hexanes until a solid precipitatedout of solution. The solvents were removed under vacuum and the solidwas triturated with hexanes:EtOAc 95:5 to give2-(pyridin-2-yldisulfanyl)propyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate62d (372 mg) as a tan solid, which was used in the next step withoutfurther purification. HRMS m/z 1003.2445 [(M+Na)⁺ calcd forC₄₈H₅₅Cl₂N₄NaO₈PS₂ 1003.2468].

TFA (3.5 mL) was added slowly to a stirred solution of 62d (prepared asabove) in DCM (7 mL) at 0° C. The mixture was stirred at thistemperature for 1 h. The solvents were then removed under vacuum at 25°C. The resulting dark green residue was dissolved in DCM and thesolution diluted with EtOAc causing a solid to precipitate out ofsolution. The solvents were removed under vacuum at 30° C. and theresidue again dissolved in DCM and diluted with EtOAc. The DCM wasremoved under vacuum to give a suspension in EtOAc. The solid wascollected by filtration and the solid then washed with EtOAc and hexanesand dried to give a green solid. This was further purified bypreparative HPLC (column: Synergi-MAX RP 4μ, 21.20×250 mm; flow rate: 12mL/min; mobile phase: solvent A: H₂O/ammonium formate buffer pH 3.5,solvent B: MeCN/H₂O 90:10; method: gradient, solvent A:solvent B 25:75to 0:100 over 19 min) to give 2-(pyridin-2-yldisulfanyl)propyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate62, 78 mg, 19% over two steps from 62c, HPLC purity: 87.4%) as a creampowder. ¹H NMR δ (400 MHz, DMSO-d₆) 9.69 (s, 1H), 8.56 (s, 1H), 8.48 (s,1H), 8.44 (d, J=4.5 Hz, 1H), 8.15 (d, J=8.3 Hz, 1H), 8.00 (d, J=8.5 Hz,1H), 7.90 (d, J=8.4 Hz, 1H), 7.85-7.76 (m, 3H), 7.55-7.48 (m, 2H),7.44-7.40 (m, 1H), 7.36-7.32 (m, 1H), 7.24-7.21 (m, 1H), 4.41-4.14 (m,8H), 4.05-3.99 (m, 2H), 3.91 (dd, J=10.8, 7.2 Hz, 1H), 3.85-3.81 (m,1H), 3.37-3.28 (m, 1H), 2.74-2.57 (m, 4H), 1.98-1.95 (m, 2H), 1.34 (d,J=6.7 Hz, 3H), 2 protons not observed. ³¹P NMR δ (400 MHz, DMSO-d₆)−5.09. HRMS m/z 891.1221 [(M+Na)⁺ calcd for C₄₀H₃₉Cl₂N₄NaO₈PS₂ 891.1216]

Example 13(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate63

(S)-1-(Chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)ethyl(methyl)carbamate57 was enzymatically dephosphorylated to give 63.

Example 14 2-(pyridin-2-yldisulfanyl)ethyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate64

2-(Pyridin-2-yldisulfanyl)ethyl(S)-1-(chloromethyl)-3-(5-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate61 was enzymatically dephosphorylated to give 64.

Example 15(11aS)-4-((S)-6-amino-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate65

To a stirred solution of (S)-2,2,2-trichloroethyl6-(5-(tert-butoxycarbonylamino)-4-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)hexanoate54c (1.66 g, 2.71 mmol) in dry DCM (10 mL) at r.t. was added aceticanhydride (1.29 mL, 13.6 mmol) and triethylamine (2.27 mL, 16.3 mmol).See FIG. 17. The reaction mixture was stirred for a further 4 h. DryMeOH (1.5 mL) was added and the mixture was stirred for 30 min. Ethylacetate (200 mL) was added and the ethyl acetate layer was separated andthen washed with water several times. The ethyl acetate solution wasdried (MgSO₄) and evaporated to give (S)-2,2,2-trichloroethyl6-(4-(2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-(tert-butoxycarbonylamino)-2-methoxyphenoxy)hexanoate65a (1.8 g, 100%) as a pale yellow glue; ¹H NMR [(CD₃)₂SO] δ 8.82 (br s,1H), 7.27 (s, 1H), 6.86 (s, 1H), 4.89 (s, 2H), 4.39-4.20 (m, 3H), 3.93(t, J=6.4 Hz, 2H), 3.74 (s, 3H), 3.50-3.33 (m, 2H), 2.10-1.94 (m, 4H),1.92-1.61 (m, 7H), 1.53-1.42 (m, 2H), 1.43 (s, 9H), 2H obscured by DMSOpeak. HRMS (ESI) m/z calc. for C₂₈H₃₉Cl₃N₂NaO₉: 675.1613. found:675.1603 [MNa⁺]. Calc. for C₂₈H₄₀Cl₃N₂O₉: 653.1794. found: 653.1778[MH⁺].

To a stirred solution of 65a (1.76 g, 2.69 mmol) in a mixture of acetone(30 mL), water (20 mL), and THF (12 mL) under nitrogen was added Zn(7.06 g, 108 mmol) and NH₄Cl (11.6 g, 216 mmol). The mixture was stirredat r.t. for 23 h. Ethyl acetate (100 mL) was added and the mixture wasstirred for 15 min. The organic layer was decanted. The extraction wasrepeated with more ethyl acetate (2×100 mL). The combined organicsolution was washed with water (2×100 mL), dried (MgSO₄), filteredthrough celite and evaporated to give(S)-6-(4-(2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-(tert-butoxycarbonylamino)-2-methoxyphenoxy)hexanoicacid 65b (1.36 g, 96%) as a sticky colorless foam; ¹H NMR [(CD₃)₂SO] δ11.49 (very br s, 1H), 8.83 (s, 1H), 7.27 (s, 1H), 6.86 (br s, 1H),4.39-4.02 (m, 3H), 3.93 (t, J=6.4 Hz, 2H), 3.74 (s, 3H), 3.51-3.33 (m,2H, partially obscured by water peak), 2.21 (t, J=7.1 Hz, 2H), 2.11-1.93(m, 4H), 1.90-1.66 (m, 5H), 1.62-1.50 (m, 2H), 1.50-1.35 (m, 2H), 1.43(s, 9H). Anal. (C₂₆H₃₈N₂O₉.) Calc: C, 59.76; H, 7.33; N, 5.36. Found: C,59.66; H, 7.49; N, 5.29.

To a stirred solution of 65b (0.87 g, 2.41 mmol) and(S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 58b (1.26 g, 2.41 mmol) in dry DMA (5mL) at 0° C. under a nitrogen atmosphere was added 4M HCl in p-dioxane(1.21 mL, 4.82 mmol), followed by EDCI.HCl (1.39 g, 7.23 mmol), andanhydrous TsOH (83 mg, 0.48 mmol). The reaction mixture was stirred at0° C. under nitrogen for 21 hours then partitioned between ethyl acetate(500 mL) and water (500 mL). The ethyl acetate layer was separated andthe aqueous layer was further extracted with more ethyl acetate (200mL). The combined ethyl acetate extracts were washed successively withwater (200 mL), saturated NaHCO₃ solution (2×200 mL) and water (200 mL).The ethyl acetate layer was dried and evaporated to give(S)-3-(6-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-(tert-butoxycarbonylamino)-2-methoxyphenoxy)hexanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 65c (1.66 g, 80%) as a beigesolid-foam; mp 84-87° C.; ¹H NMR [(CD₃)₂SO] δ 8.84 (br s, 1H), 8.21 (s,1H), 7.95 (d, J=8.3 Hz, 1H), 7.81 (d, J=8.3 Hz, 1H), 7.58 (br t, J=7.7,1H), 7.46 (br t, J=8.1 Hz, 1H), 7.29 (s, 1H), 6.86 (s, 1H), 4.40 (t,J=10.0 Hz, 1H), 4.36-3.86 (m, 10H), 3.83-3.74 (m, 1H), 3.73 (s, 3H),3.54-3.36 (m, 4H), 2.67-2.34 (m, 6H, partially obscured by DMSO peak),2.26 (s, 3H), 2.02 (br s, 3H), 1.93-1.62 (m, 8H), 1.60-1.47 (m, 2H),1.42 (s, 9H). Anal. (C₄₅H₅₈ClN₅O₁₀.1½H₂O) Calc: C, 60.63; H, 6.90; N,7.86. Found: C, 60.39; H, 6.66; N, 8.08.

To a stirred solution of 65c (2.17 g, 2.51 mmol) in DCM (20 mL) at 0° C.under a nitrogen atmosphere was added TFA (20 mL). After addition, themixture was stirred further at this temperature for 2.5 h. The mixturewas poured into a cold (0° C.) mixture of NaHCO₃ (50 g), water (700 mL),and DCM (500 mL) and stirred for 15 min. (pH ca. 8). The DCM layer wasseparated and washed with more aqueous NaHCO₃ (200 mL) and water (200mL) and then dried (MgSO₄). The solvent was evaporated to give(S)-3-(6-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-amino-2-methoxyphenoxy)hexanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 65d as a pale brown solid-foam (1.76 g,92%); mp 62° C.; ¹H NMR [(CD₃)₂SO] δ 8.21 (s, 1H), 7.95 (d, J=8.3 Hz,1H), 7.81 (d, J=8.3 Hz, 1H), 7.57 (br t, J=7.6 Hz, 1H), 7.46 (br t,J=7.2 Hz, 1H), 6.67 (s, 1H), 6.37 (s, 1H), 5.09 (s, 2H), 4.41 (t, J=9.7Hz, 1H), 4.36-4.20 (m, 3H), 4.17-4.00 (m, 3H), 3.97-3.86 (m, 3H),3.81-3.70 (m, 2H), 3.63 (s, 3H), 3.54-3.32 (m, 5H), 2.66-2.34 (m, 6H,partially obscured by DMSO peak), 2.26 (s, 3H), 2.08-1.96 (m, 1H), 2.10(s, 3H), 1.93-1.63 (m, 7H), 1.57-1.45 (m, 2H). Anal. (C₄₀H₅₀ClN₅O₈.½H₂O)Calc: C, 62.13; H, 6.65; N, 9.06. Found: C, 62.12; H, 6.76; N, 8.77.

A mixture of(S)-2-(allyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanoic acid65e (3.30 g, 10.0 mmol) and EEDQ (3.71 g, 15.0 mmol) in dry DMA (10 mL)was stirred at r.t. under nitrogen for 15 min. See FIG. 18. To thispreformed mixture was added a solution of4-((tert-butyldimethylsilyloxy)methyl)aniline (prepared from thecorresponding p-nitrobenzyl alcohol and TBDMSCl in DMF; followed byreduction using Zn/NH₄Cl) (2.37 g, 10.0 mmol) in dry DMA (3 mL). Thefinal reaction mixture was stirred further at r.t. under a nitrogenatmosphere for 23 h. The mixture was partitioned between ethyl acetate(500 mL) and water (500 mL). The ethyl acetate layer was separated andwashed successively with saturated NaHCO₃ (2×300 mL) and water (300 mL)and then dried (MgSO₄). Evaporation of the solvent gave an orange oilwhich was purified by a silica column chromatography (petroleumether-ethyl acetate gradient from 10-35%) to afford the TBDMS-protectedlysine 65f (4.87 g, 89%) as a sticky beige solid-foam; ¹H NMR [(CD₃)₂SO]δ 9.97 (s, 1H), 7.55 (d, J=8.50 Hz, 2H), 7.44 (d, J=7.8 Hz, 1H), 7.21(d, J=8.5 Hz, 2H), 6.75 (t, J=5.3 Hz, 1H), 5.99-5.82 (m, 1H), 5.28 (brd, J=17.2 Hz, 1H), 5.17 (br d, J=10.5 Hz, 1H), 4.64 (s, 2H), 4.46 (d,J=5.2 Hz, 2H), 4.12-4.02 (m, 1H), 2.93-2.83 (m, 2H), 1.70-1.52 (m, 2H),1.46-1.20 (m, 4H), 1.35 (s, 9H), 0.89 (s, 9H), 0.06 (s, 6H). HRMS (ESI)m/z calc. for C₂₈H₄₇N₃NaO₆Si: 572.3126. found: 572.3136 [MNa⁺].

To a stirred solution of 65f (4.81 g, 8.75 mmol) in THF (30 mL) at r.t.was added a 1M solution of tetrabutylammonium fluoride in THF (17.5 mL,17.5 mmol). After addition, the mixture was stirred at this temperaturefor a further 2.5 h. Aqueous NH₄Cl (300 mL) was added and product wasextracted into ethyl acetate (500 mL). The ethyl acetate was washed withwater (2×100 mL) and dried (MgSO₄). The solvent was evaporated to givebenzyl alcohol lysine 65g (3.81 g, 100%) as a beige solid; mp 101-103°C.; ¹H NMR [(CD₃)₂SO] δ 9.94 (s, 1H), 7.52 (d, J=8.4 Hz, 2H), 7.44 (d,J=7.8 Hz, 1H), 7.23 (d, J=8.4 Hz, 2H), 6.76 (t, J=5.4 Hz, 1H), 5.97-5.84(m, 1H), 5.29 (br d, J=17.2 Hz, 1H), 5.17 (br d, J=10.4 Hz, 1H), 5.08(t, J=5.7 Hz, 1H), 4.47 (d, J=5.3 Hz, 2H), 4.43 (d, J=5.7 Hz, 2H),4.13-4.03 (m, 1H), 2.96-2.82 (m, 2H), 1.72-1.52 (m, 2H), 1.46-1.20 (m,4H), 1.36 (s, 9H). HRMS (ESI) m/z calc. for C₂₂H₃₃N₃NaO₆: 458.2262.found: 458.2272 [MNa⁺]; calc. for C₂₂H₃₃N₃KO₆: 474.2001. found: 474.1998[MK⁺].

To a stirred solution of 65d (764 mg, 1.00 mmol) and DMAP (367 mg, 3.00mmol) in dry DCM (15 mL) at r.t. under nitrogen was added a solution ofdiphosgene in dry DCM (0.05 mmol per mL, 12 mL, 0.60 mmol) and themixture was stirred for a further 20 min. See FIG. 19. To this mixturewas added a solution of 65g (3.97 g, 9.13 mmol) in dry DCM (80 mL). Thefinal reaction mixture was stirred further at r.t. under a nitrogenatmosphere for 48 h. The mixture was partitioned between ethyl acetate(500 mL) and water (300 mL). The ethyl acetate layer was separated andthe aqueous layer was further extracted with ethyl acetate (2×200 mL).The combined ethyl acetate solution was washed with more water (2×200mL) and dried (MgSO₄). Evaporation of the solvent at 30° C. (bathtemperature) gave an orange oil which was purified by silica columnchromatography (ethyl acetate-MeOH=10:1) to afford(S)-3-(6-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-((4-((S)-2-(allyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanamido)benzyloxy)carbonylamino)-2-methoxyphenoxy)hexanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 65h (1.04 g, 85%) as a pale orangesolid; mp 90-93° C.; ¹H NMR [(CD₃)₂SO] δ 10.04 (s, 1H), 9.10 (br s, 1H),8.21 (s, 1H), 7.95 (d, J=8.3 Hz, 1H), 7.81 (d, J=8.3 Hz, 1H), 7.63-7.53(m, 3H), 7.51-7.42 (m, 2H), 7.32 (d, J=8.5 Hz, 2H), 7.21 (br s, 1H),6.85 (br s, 1H), 6.79-6.72 (m, 1H), 5.97-5.83 (m, 1H), 5.29 (br d,J=17.2 Hz, 1H), 5.17 (br d, J=10.4 Hz, 1H), 5.08-4.96 (m, 2H), 4.52-4.37(m, 3H), 4.37-3.85 (m, 10H), 3.83-3.66 (m, 2H), 3.74 (s, 3H), 3.54-3.41(m, 2H), 3.41-3.23 (m, 2H, partially obscured by water peak), 2.95-2.83(m, 2H), 2.66-2.34 (m, 6H, partially obscured by DMSO peak), 2.25 (s,3H), 2.07-1.92 (m, 4H), 1.87-1.45 (m, 11H), 1.45-1.20 (m, 4H), 1.35 (s,9H). HRMS (ESI) m/z calc. for C₆₃H₈₂ClN₈O₁₅: 1225.5583. found: 1225.5557[MH⁺]; calc. for C₆₃H₈₁ClN₈NaO₁₅: 1247.5402. found: 1247.5401 [MNa⁺];calc. for C₆₃H₈₁ClKN₈O₁₅: 1263.5142. found: 1263.5141 [MK⁺].

A mixture of 65h (1.01 g, 0.824 mmol) and K₂CO₃ (1.14 g, 8.24 mmol) inDCM (20 mL) and MeOH (10 mL) was stirred at r.t. for 1 hour and 40 min.The mixture was diluted with DCM (200 mL) and stirred with ice-water(200 mL) for 10 min. The DCM layer was separated and the aqueous layerwas further extracted with DCM (2×100 mL). The combined DCM solution waswashed with more water (200 mL) and dried (MgSO₄). Evaporation ofsolvent at 25° C. (bath temperature) gave(S)-3-(6-(5-((4-((S)-2-(allyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanamido)benzyloxy)carbonylamino)-4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)hexanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl4-methylpiperazine-1-carboxylate 65i (0.94 g, 96%) as a beige solid; mp104-107° C.; ¹H NMR [(CD₃)₂SO] δ 10.04 (s, 1H), 9.17 (br s, 1H), 8.21(s, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.80 (d, J=8.3 Hz, 1H), 7.63-7.53 (m,3H), 7.51-7.42 (m, 2H), 7.38-7.21 (m, 3H), 6.93 (s, 1H), 5.32 (t, J=5.4Hz, 1H), 5.98-5.83 (m, 1H), 5.30 (br d, J=17.2 Hz, 1H), 5.17 (br d,J=11.7 Hz, 1H), 5.03 (s, 2H), 4.73 (t, J=5.7 Hz, 1H), 4.52-4.36 (m, 3H),4.36-4.17 (m, 2H), 4.17-3.85 (m, 6H), 3.83-3.66 (m, 2H), 3.73 (s, 3H),3.61-3.40 (m, 4H), 3.40-3.20 (m, 2H, partially obscured by water peak),2.94-2.83 (m, 2H), 2.67-2.34 (m, 6H, partially obscured by DMSO peak),2.25 (s, 3H), 1.96-1.45 (m, 12H), 1.45-1.20 (m, 4H), 1.35 (s, 9H). HRMS(ESI) m/z calc. for C₆₁H₈₀ClN₈O₁₄: 1183.5477. found: 1183.5445 [MH⁺];calc. for C₆₁H₇₉ClN₈NaO₁₄: 1205.5296. found: 1205.5256 [MNa⁺]; calc. forC₆₁H₇₉ClKN₈O₁₄: 1221.5036. found: 1221.5026 [MK⁺].

To a stirred solution of 65i (0.92 g, 0.78 mmol) in dry DCM (20 mL) at0° C. was added Dess-Martin periodinane (DMP,1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one, CAS Reg. No.87413-09-0, 492 mg, 1.16 mmol) portionwise (over 8 min). After additionwas complete the reaction mixture was stirred further at 0° C. for 2 h,then at r.t. for 45 h. The mixture was diluted with DCM (100 mL) andstirred with 10% Na₂S₂O₃ (100 mL) at r.t. for 10 min. The resultingmixture was partitioned between DCM (400 mL) and saturated NaHCO₃solution (400 mL). The DCM layer was separated and the aqueous layer wasfurther extracted with DCM (2×100 mL). The combined DCM solution wasfurther washed with saturated NaHCO₃ solution (200 mL) and water (200mL) and then dried (MgSO₄). Evaporation of solvent at 25° C. (bathtemperature) gave a pale brown solid which was purified by SiO₂ columnchromatography (DCM-ethyl acetate-MeOH=15:15:1, gradient to 15:15:3) togive(S)-4-((S)-2-(allyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate65j (0.64g, 70%) as a pale yellow solid; mp 137° C. (dec.); ¹H NMR[(CD₃)₂SO] δ 10.02 (s, 1H), 8.21 (s, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.80(d, J=8.3 Hz, 1H), 7.65-7.38 (m, 5H), 7.18 (d, J=7.0 Hz, 2H), 7.03 (s,1H), 6.82-6.63 (m, 2H), 6.49 (poorly resolved d, J=4.7 Hz, exchangeablewith D₂O, 1H), 5.96-5.82 (m, 1H), 5.46 (poorly resolved dd, J=9.8, 4.7Hz, became a d after D₂O, J=10.1 Hz, 1H), 5.27 (br d, J=17.1 Hz, 1H),5.21-5.10 (m, 2H), 4.81 (br d, J=12.3 Hz, 1H), 4.51-4.17 (m, 5H),4.13-3.84 (m, 4H), 3.84-3.67 (m, 2H), 3.77 (s, 3H), 3.55-3.20 (m, 6H,partially obscured by water peak), 2.66-2.30 (m, 6H, partially obscuredby DMSO peak), 2.26 (s, 3H), 2.10-1.20 (m, 16H), 1.35 (s, 9H). HRMS(ESI) m/z calc. for C₆₁H₇₈ClN₈O₁₄:1181.5321. found: 1181.5286 [MH⁺];calc. for C₆₁H₇₇ClN₈NaO₁₄: 1203.5140. found: 1203.5130 [MNa⁺]; calc. forC₆₁H₇₇ClKN₈O₁₄: 1219.4879. found: 1219.4861 [MK⁺].

To a stirred solution of 65j (623 mg, 0.53 mmol) in dry DCM (15 mL) at0° C. under a nitrogen atmosphere was added pyrrolidine (0.86 mL, 10.5mmol), followed by Pd(Ph₃P)₄ (30 mg, 9.8% Pd). After addition thereaction mixture was stirred further at r.t. for 5 h. The mixture wasdiluted with petroleum ether (100 mL) and stirred at r.t. for 30 min.The solvents were decanted from the insoluble material and the wash stepwas repeated with DCM-petroleum ether (1:10) (100 mL). The sticky solidleft behind was dissolved in DCM (200 mL) and washed with water (3×100mL), then dried (MgSO₄), and passed through a short SiO₂ column toremove base-line material. The required compound was eluted using aDCM-MeOH gradient (from 2 to 5%). Evaporation of the solvent at 25° C.(bath temperature) gave a pale yellow solid-foam (472 mg, 82%) which wasused directly in the next step. This crude amine was treated withFmoc-val-Osu (N-α-Fmoc-L-valine N-hydroxysuccinimide ester, 550 mg, 1.26mmol) in dry DMA (8 mL) at r.t. under a nitrogen atmosphere and themixture was stirred for 5 h. Ethyl acetate-petroleum ether (1:10, 100mL) was added and the mixture was stirred at r.t. for 20 min. Thesolvent was decanted from the insoluble material and the wash step wasrepeated with more ethyl acetate-petroleum ether (1:10, 2×50 mL). Thesticky solid left behind was dried and purified by SiO₂ columnchromatography (DCM-ethyl acetate-MeOH=20:10:3) to give(S)-4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbutanamido)-6-(tert-butoxycarbonylamino)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate65k (246 mg, 47%) as a colorless solid; mp 143° C. (dec.); ¹H NMR[(CD₃)₂SO] δ 10.03 (s, exchangeable with D₂O, 1H), 8.21 (s, 1H), 8.05(br d, J=7.5 Hz, exchangeable with D₂O, 1H), 7.95 (d, J=8.5 Hz, 1H),7.87 (d, J=7.4 Hz, 2H), 7.81 (d, J=8.3 Hz, 1H), 7.72 (t, J=7.0 Hz, 2H),7.62-7.25 (m, 10H, reduced to 9H after D₂O), 7.18 (poorly resolved d,J=5.8 Hz, 2H), 7.04 (s, 1H), 6.70 (br s, 2H, reduced to 1H after D₂O),6.50 (br s, exchangeable with D₂O, 1H), 5.53-5.40 (m, became a d afterD₂O, J=9.9 Hz, 1H), 5.15 (br d, J=12.4 Hz, 1H), 4.82 (br d, J=12.3 Hz,1H), 4.46-4.16 (m, 7H), 4.07-3.85 (m, 4H), 3.83-3.67 (m, 2H), 3.76 (s,3H), 3.56-3.23 (m, 5H, partially obscured by water peak), 2.93-2.79 (m,2H), 2.64-2.32 (m, 6H, partially obscured by DMSO peak), 2.25 (s, 3H),2.09-1.20 (m, 17H), 1.35 (s, 9H), 0.87 (d, J=7.0 Hz, 3H), 0.83 (d, J=6.8Hz, 3H). HRMS (ESI) m/z calc. for C₇₇H₉₃ClN₉O₁₅:1418.6474. found:1418.6420 [MH⁺]; calc. for C₇₇H₉₂ClN₉NaO₁₅: 1440.6294. found: 1440.6231[MNa⁺]; calc. for C₇₇H₉₂ClKN₉O₁₅: 1456.6033. found: 1456.6021 [MK⁺].

To a stirred solution of 65k (224 mg, 0.158 mmol) in dry DMA (2 mL) at0° C. under a nitrogen atmosphere was added a solution of piperidine inN,N-dimethylacetamide (DMA, 1.0 mmol per mL, 1.58 mL, 1.58 mmol). SeeFIG. 20. After addition the mixture was stirred at this temperature fora further 1 h and 45 min. A mixture of ethyl acetate-petroleum ether(1:5, 50 mL) was added and the mixture stirred at 0° C. for 20 min. Thesolvent layer was decanted from the insoluble material and discarded.The wash step was repeated with more ethyl acetate-petroleum ether (1:5,2×30 mL) at r.t. The pale yellow solid left behind was dried to give(S)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-6-(tert-butoxycarbonylamino)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate651 (173 mg, 99%); mp 74° C. (dec.); ¹H NMR [(CD₃)₂SO] δ 10.09 (s,exchangeable with D₂O, 1H), 8.21 (s, 1H), 8.08 (br s, exchangeable withD₂O, 1H), 7.96 (d, J=8.4 Hz, 1H), 7.81 (d, J=8.2 Hz, 1H), 7.66-7.42 (m,4H), 7.19 (poorly resolved d, J=7.5 Hz, 2H), 7.04 (s, 1H), 6.71 (br s,2H, reduced to 1H after D₂O), 6.49 (br s, exchangeable with D₂O, 1H),5.52-5.40 (m, became a d after D₂O, J=10.6 Hz, 1H), 5.16 (br d, J=12.1Hz, 1H), 4.81 (br d, J=11.7 Hz, 1H), 4.49-4.18 (m, 4H), 4.10-3.85 (m,3H), 3.85-3.67 (m, 2H), 3.77 (s, 3H), 3.59-3.22 (m, 5H, partiallyobscured by water peak), 3.03-2.97 (m, became a d after D₂O, J=4.8 Hz,1H), 2.91-2.81 (m, 2H), 2.71-2.33 (m, 7H, partially obscured by DMSOpeak), 2.25 (s, 3H), 2.11-1.20 (m, 17H), 1.34 (s, 9H), 0.86 (d, J=6.5Hz, 3H), 0.76 (d, J=6.7 Hz, 3H), 2H not observed. HRMS (ESI) m/z calc.for C₆₂H₈₃ClN₉O₁₃:1196.5793. found: 1196.5804 [MH⁺]; calc. forC₆₂H₈₂ClN₉NaO₁₃: 1218.5613. found: 1218.5612 [MNa⁺]; calc. forC₆₂H₈₂ClKN₉O₁₃: 1234.5352. found: 1234.5359 [MK⁺].

A mixture of 65l (173 mg, 0.158 mmol) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (maleimido-Osu, 122mg, 0.395 mmol) in dry DMA (2 mL) was stirred at 0° C. under a nitrogenatmosphere for 1 h 45 min. A mixture of ethyl acetate-petroleum ether(1:5, 50 mL) was added and the resulting mixture stirred at 0° C. for 15min. The solvent layer was decanted from the insoluble material anddiscarded. The wash step was repeated with more ethyl acetate-petroleumether (1:5, 2×30 mL) at r.t. The solid left behind was dried andpurified by silica column chromatography (DCM:ethylacetate:MeOH=20:10:3) to give(S)-4-((S)-6-(tert-butoxycarbonylamino)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate65m (152 mg, 69%) as a pale yellow solid; HPLC: 90.6% pure; mp 120° C.;¹H NMR [(CD₃)₂SO] δ 9.95 (s, exchangeable with D₂O, 1H), 8.21 (s, 1H),8.01 (d, J 7.4 Hz, exchangeable with D₂O, 1H), 7.95 (d, J=8.4 Hz, 1H),7.85-7.73 (m, 2H, reduced to 1H after D₂O exchange), 7.63-7.50 (m, 3H),7.46 (t, J=7.7 Hz, 1H), 7.17 (poorly resolved d, J=8.4 Hz, 2H), 7.04 (s,1H), 6.98 (s, 2H), 6.81-6.66 (m, 2H, reduced to 1H after D₂O exchange),6.49 (poorly resolved d, J=5.3 Hz, exchangeable with D₂O, 1H), 5.51-5.41(m, became a d after D₂O, J=9.9 Hz, 1H), 5.15 (d, J=12.6 Hz, 1H), 4.81(br d, J=11.3 Hz, 1H), 4.41 (t, J=9.7 Hz, 1H), 4.37-4.27 (m, 2H),4.27-4.13 (m, 2H), 4.10-3.85 (m, 3H), 3.85-3.63 (m, 2H), 3.77 (s, 3H),3.60-3.21 (m, 8H, partially obscured by water peak, 1H), 2.92-2.81 (m,2H), 2.67-2.33 (m, 6H, partially obscured by DMSO peak), 2.26 (s, 3H),2.23-1.11 (m, 25H), 1.30 (s, 9H), 0.84 (d, J=6.8 Hz, 3H), 0.81 (d, J=6.7Hz, 3H). HRMS (ESI) m/z calc. for C₇₂H₉₄ClN₁₀O₁₆:1389.6532. found:1389.6478 [MH⁺]; calc. for C₇₂H₉₄ClN₁₀NaO₁₆: 706.3212. found: 706.3244[MH⁺Na⁺]; calc. for C₇₂H₉₃ClN₁₀Na₂O₁₆: 717.3122. found: 717.3121[MNa⁺Na⁺].

To a stirred solution of 65m (32.2 mg, 0.023 mmol) in DCM (0.4 mL) at 0°C. (bath temperature) under nitrogen was added TFA (0.8 mL), followed bya solution of 2% water in TFA (0.8 mL). After addition the mixture wasstirred at this temperature for a further 8 h. Ethyl acetate-petroleumether (1:5, 25 mL) was added and the mixture was stirred at 0° C. for 15min. The precipitated solid was collected, washed with ethylacetate-petroleum ether (1:5, 2×30 ml), and dried to give a crudeproduct (30 mg) which was purified by preparative HPLC [SynergiMaxRPcolumn; water-TFA (pH=2.56; 95% to 55%)/10% H₂O in CH₃CN (5% to 45%);flow rate: 12 mL/min] to give(S)-4-((S)-6-amino-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)hexanamido)benzyl8-(6-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyloxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate65 as the bis-trifluoroacetate salt (22.2 mg, 64%) as a glassy solid;HPLC: 97.1% pure; mp 114° C.; [α]_(D)+43.6° (c 0.275, MeOH); ¹H NMR[(CD₃)₂SO] δ 9.97 (s, 1H), 9.90 (br s, 1H), 8.73 (br s, 2H), 8.27 (s,1H), 8.09 (d, J=7.6 Hz, 1H), 8.04-7.93 (m, 2H), 7.89 (d, J=8.4 Hz, 1H),7.82 (d, J=7.9 Hz, 1H), 7.74-7.50 (m, 5H), 7.46 (br t, J=7.6 Hz, 1H),7.22 (m, 2H), 7.04 (s, 1H), 7.00 (s, 2H), 6.75 (s, 1H), 6.51 (br s, 1H),5.52-5.40 (m, 1H), 5.18-5.04 (m, 1H), 4.94-4.82 (m, 1H), 4.48-3.70 (m,14H), 3.43-3.20 (m, 4H, partially obscured by water peak), 3.17-3.05 (m,3H), 2.89 (s, 3H), 2.82-2.70 (m, 2H), 2.66-2.48 (m, 2H, partiallyobscured by DMSO peak), 2.25-1.08 (m, 25H), 0.91-0.77 (m, 6H), 2H notobserved. HRMS (ESI) m/z calc. for C₆₇H₈₆ClN₁₀O₁₄:1289.6008. found:1289.5975 [MH⁺]; calc. for C₆₇H₈₅ClN₁₀NaO₁₄: 1311.5827. found: 1311.5772[MNa⁺]; calc. for C₆₇H₈₅ClN₁₀Na₂O₁₄: 667.2860. found: 667.2874[MNa⁺Na⁺]; calc. for C₆₇H₈₆ClN₁₀NaO₁₄:656.2950. found: 656.2963[MH⁺Na⁺]; calc. for C₆₇H₈₇ClN₁₀O₁₄: 645.3040. found: 645.3052 [MH⁺H⁺].

Example 16N-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-phosphonoxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide66

To a solution of (S)-tert-butyl5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate 57a,prepared from 51a in Example 7 (1.595 g, 3.76 mmol) in DCM (15 mL)cooled in an ice bath was added 4N HCl in dioxane (40 mL). See FIG. 21.The mixture was allowed to warm up to room temperature and stirred for 2h. All volatile components were pumped off. The resultant residue wasredistributed between ethyl acetate and cold aqueous 5% ammonia. Theaqueous phase was extracted with ethyl acetate three times. The combinedorganic extracts were washed with water followed by brine, dried overanhydrous Na₂SO₄, and filtered through celite. The solvent was removedto give (S)-5-(benzyloxy)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole57b as a brown gum, which was used directly; ¹H NMR (DMSO) δ 8.04 (d,J=8.2 Hz, 1H), 7.61 (d, J=8.3 Hz, 1H), 7.53 (d, J=7.2 Hz, 2H), 7.45-7.34(m, 4H), 7.14 (t, J=7.3 Hz, 1H), 6.60 (s, 1H), 5.24 (s, 2H), 3.96-3.92(m, 1H), 3.84 (dd, J=3.4, 10.7 Hz, 1H), 3.70 (t, J=9.3 Hz, 1H), 3.60(dd, J=2.4, 10.0 Hz, 1H), 3.55 (t, J=10.3 Hz, 1H) ppm. HRMS (ESI) foundm/z 324.1150 (M+H). C₂₀H₁₉ClNO requires 324.1150.

Intermediate 57b was cooled in an ice bath and pyridine (15 mL) wasadded, followed by trifluoroacetic anhydride (3.14 mL, 22.57 mmol). Theresultant mixture was stirred for 10 min and ice was added. The mixturewas redistributed between ethyl acetate and water. The aqueous phase wasextracted with ethyl acetate three times. The combined organic extractswere washed with water followed by brine, dried over anhydrous Na₂SO₄,and filtered through celite. The solvent was removed and the resultantresidue was purified by column chromatography using a mixture of ethylacetate and petroleum ether (v/v 1:10) as eluent to give(S)-1-(5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-2,2,2-trifluoroethanone66a as a white solid (1.11 g, 70%); mp 167-170° C. ¹H NMR (CDCl₃) δ 8.37(d, J=8.3 Hz, 1H), 8.05 (s, 1H), 7.72 (d, J=8.2 Hz, 1H), 7.61-7.54 (m,3H), 7.49-7.42 (m, 3H), 7.39-7.35 (m, 1H), 5.30 (AB q, J=11.7, 15.7 Hz,2H), 4.63-4.59 (m, 1H), 4.43-4.38 (m, 1H), 4.15-4.09 (m, 1H), 3.97-3.93(m, 1H), 3.49 (dd, J=9.9, 11.3 Hz, 1H) ppm. HRMS (ESI) found m/z442.0799 (M+Na). C₂₂H₁₇ClF₃NNaO₂ requires 442.0795.

At −10° C., to a solution of 66a (1.10 g, 2.62 mmol) in THF (20 mL) wasadded 25% aqueous ammonium formate (20 mL) followed by Pd—C catalyst(10%, wet, 550 mg) and the mixture was stirred for 2 h before more Pd—Ccatalyst (550 mg) was added. The resultant mixture was stirred at −10°C. overnight and the catalyst was filtered off through celite. THF wasremoved from the filtrate and the residue was redistributed betweenethyl acetate and water. The aqueous phase was extracted with ethylacetate three times. The combined organic extracts were washed withwater followed by brine, dried over anhydrous Na₂SO₄, and filteredthrough celite. The solvent was removed and the resultant residue waspurified by column chromatography using a mixture of ethyl acetate andpetroleum ether (v/v 1:5) as eluent to give(S)-1-(1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-2,2,2-trifluoroethanone66b as an off-white solid (758 mg, 88%); mp 209-212° C. ¹H NMR (CDCl₃) δ8.33 (d, J=8.2 Hz, 1H), 8.10 (s, 1H), 7.85 (s, 1H), 7.64 (d, J=8.2 Hz,1H), 7.60-7.56 (m, 1H), 7.51-7.47 (m, 1H), 4.60-4.56 (m, 1H), 4.41-4.36(m, 1H), 4.00-3.95 (m, 1H), 3.93-3.90 (m, 1H), 3.44 (dd, J=9.8, 11.3 Hz,1H) ppm. HRMS (ESI) found m/z 352.0331 (M+Na). C₁₅H₁₁ClF₃NNaO₂ requires352.0323.

To a solution of 66b (250 mg, 0.76 mmol) in THF (15 mL) was addedtetrazole (3% in acetonitrile, 13.5 mL, 4.55 mmol) followed bydi-tert-butyl-N,N-di-isopropyl phosphoramidite (1.51 mL, 4.55 mmol). Themixture was stirred at room temperature overnight then cooled in an icebath and H₂O₂(30% aqueous solution, 0.78 mL, 7.58 mmol) was addeddropwise. The resultant mixture was allowed to warm up to roomtemperature and stirred for 5 h. The reaction was quenched by theaddition of 10% aqueous sodium sulphite with cooling in an ice bath.Organic volatiles were removed by rotary evaporator. The resultantmixture was redistributed between ethyl acetate and water. The aqueousphase was extracted with ethyl acetate three times. The combined organicextracts were washed with water followed by brine, dried over anhydrousNa₂SO₄, and filtered through celite. The solvent was removed and theresultant residue was purified by Florisil® (US Silica) columnchromatography using gradient mixtures of ethyl acetate and petroleumether (v/v 1:6 to 1:3) as eluent to give (S)-di-tert-butyl1-(chloromethyl)-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-5-ylphosphate 66c as colorless oil (367 mg, 93%); ¹H NMR (DMSO) δ 8.44 (d,J=1.0 Hz, 1H), 8.11 (d, J=8.1 Hz, 1H), 8.06 (d, J=8.2 Hz, 1H), 7.69-7.65(m, 1H), 7.63-7.59 (m, 1H), 4.61-4.56 (m, 1H), 4.46-4.41 (m, 1H),4.15-4.12 (m, 1H), 4.06-4.00 (m, 1H), 1.50 (s, 9H), 1.48 (s, 9H) ppm.³¹P NMR (DMSO) δ −15.54 ppm. HRMS (ESI) found m/z 544.1236 (M+Na).C₂₃H₂₈ClF₃NNaO₅P requires 544.1238.

To a solution of 66c (239 mg, 0.46 mmol) in MeOH (2 mL) cooled in an icebath was added CsCO₃ (298 mg, 0.92 mmol) and several drops of water. Themixture was stirred in the ice bath for 1 h and then redistributedbetween ethyl acetate and water. The aqueous phase was extracted withethyl acetate three times. The combined organic extracts were washedwith water and brine, dried over anhydrous Na₂SO₄, filtered throughcelite, and the solvent was removed. The resultant residue was dissolvedin ethyl acetate and filtered through a pad of Florisil® (US Silica)column chromatography to give (S)-di-tert-butyl1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl phosphate 66d as anoff-white gum (183 mg, 94%) which was used directly without furtherpurification; ¹H NMR (DMSO) δ 8.08 (d, J=8.4 Hz, 1H), 7.58 (d, J=8.3 Hz,1H), 7.46-7.42 (m, 1H), 7.25-7.21 (m, 1H), 7.13 (d, J=0.8 Hz, 1H),4.00-3.93 (m, 1H), 3.87-3.78 (m, 2H), 3.54-3.42 (m, 2H), 1.50 (s, 9H),1.49 (s, 9H) ppm. ³¹P NMR (DMSO) δ −15.58 ppm. HRMS (ESI) found m/z426.1587 (M+H). C₂₁H₃₀ClNO₄P requires 426.1595.

2,5-Dibromonitrobenzene (5.0 g, 17.8 mmol) and t-butyl acrylate (7.75mL, 53.40 mmol) were dissolved in triethylamine (50 mL). See FIG. 22.The flask was flushed by bubbling nitrogen gas through the solution,then tri-p-tolyl phosphine (433 mg, 1.42 mmol) and palladium acetate (80mg, 0.36 mmol) were added under nitrogen flow. The mixture was stirredat reflux overnight under nitrogen. Triethylamine was pumped off. Theresultant residue was dissolved in ethyl acetate and filtered through apad of silica gel. The filtrate was concentrated and loaded on achromatography column. A mixture of ethyl acetate and petroleum ether(1:10) was used as eluent to give (2E,2′E)-tert-butyl3,3′-(2-nitro-1,4-phenylene)diacrylate 66e as a white solid (5.85 g,88%); mp 123-124° C. ¹H NMR (CDCl₃) δ 8.14 (d, J=1.7 Hz, 1H), 7.99 (d,J=15.8 Hz, 1H), 7.74 (dd, J=1.7, 8.2 Hz, 1H), 7.66 (d, J=8.1 Hz, 1H),7.58 (d, J=16.0 Hz, 1H), 6.50 (d, J=16.0 Hz, 1H), 6.36 (d, J=15.8 Hz,1H), 1.58 (s, 9H), 1.56 (s, 9H) ppm. HRMS (ESI) found m/z 398.1574(M+Na). C₂₀H₂₅NNaO₆ requires 398.1574.

To a solution of 66e (5.85 g, 15.58 mmol) in acetone (40 mL) cooled inan ice bath was added zinc powder (8.15 g, 125.0 mmol), followed by asolution of NH₄Cl (3.33 g, 62.30 mmol) in water (20 mL). The mixture wasstirred at room temperature for 1 h. More zinc powder (4.00 g) and moreNH₄Cl (1.7 g) in water (10 mL) were added. After 1 h, ethyl acetate (100mL) was added and the upper clear solution was collected by decanting.The wash and decanting steps were repeated two more times. The combinedorganic solution was washed with water followed by brine, dried overanhydrous Na₂SO₄, and filtered through a pad of silica gel. The solventwas removed to give (2E,2′E)-tert-butyl3,3′-(2-amino-1,4-phenylene)diacrylate 66f as a yellow gum (5.46 g,100%); mp 73-75° C. ¹H NMR (CDCl₃) δ 7.68 (d, J=15.8 Hz, 1H), 7.46 (d,J=16.0 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 6.92 (dd, J=1.5, 8.1 Hz, 1H),6.81 (d, J=1.4 Hz, 1H), 6.33 (d, J=16.0 Hz, 1H), 6.31 (d, J=15.8 Hz,1H), 3.99 (br s, 2H), 1.53 (s, 9H), 1.51 (s, 9H) ppm. HRMS (ESI) foundm/z 368.1830 (M+Na). C₂₀H₂₇NNaO₄ requires 368.1832.

A mixture of 66f (2.73 g, 7.90 mmol),3-(((9H-fluoren-9-yl)methoxy)carbonylamino)propanoic acid(Fmoc-beta-alanine, 3.69 g, 11.85 mmol), EDCI hydrochloride (7.58 g,39.50 mmol) and p-toluenesulfonic acid (136 mg, 0.79 mmol) in DMA (25mL) was stirred at room temperature overnight. The mixture wasredistributed between ethyl acetate and water. The aqueous phase wasextracted with ethyl acetate three times. The combined organic extractswere washed with water followed by brine, dried over anhydrous Na₂SO₄,and filtered through celite. The solvent was removed to give(2E,2′E)-tert-butyl3,3′-(2-(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)propanamido)-1,4-phenylene)diacrylate66g as a white solid (4.89 g, 97%); mp 102-105° C. ¹H NMR (CDCl₃) δ 7.87(s, 1H), 7.70-7.66 (m, 2H), 7.62-7.52 (m, 4H), 7.44 (br s, 1H),7.38-7.34 (m, 3H), 7.29-7.25 (m, 2H), 6.41 (d, J=16.0 Hz, 1H), 6.34 (d,J=15.7 Hz, 1H), 4.42 (br s, 2H), 4.19 (t, J=6.4 Hz, 1H), 3.59 (br s,2H), 2.67 (br s, 2H), 1.53 (s, 9H), 1.51 (s, 9H) ppm. HRMS (ESI) foundm/z 661.2878 (M+Na). C₃₈H₄₂N₂NaO₇ requires 661.2884.

To a solution of 66g (530 mg, 0.83 mmol) in DCM (4 mL) cooled in an icebath was added TFA (1 mL, 12.98 mmol). The mixture was allowed to warmup to room temperature and stirred overnight to give a white suspension.Ethyl acetate was added to precipitate out more solid, which wascollected by filtration and washed with ethyl acetate and petroleumether, to give(2E,2′E)-3,3′-(2-(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)propanamido)-1,4-phenylene)diacrylicacid 66h as a white solid (404 mg, 92%). mp 282° C. (dec.). ¹H NMR(DMSO) δ 12.46 (br s, 1H), 9.92 (s, 1H), 7.89-7.84 (m, 3H), 7.74-7.68(m, 4H), 7.58-7.54 (m, 2H), 7.44-7.38 (m, 3H), 7.31-7.28 (m, 2H), 6.54(dd, J=3.2, 16.0 Hz, 2H), 4.30 (d, J=6.5 Hz, 2H), 4.22 (t, J=6.8 Hz,1H), 3.30 (br s, 2H), 2.83-2.79 (m, 2H) ppm. HRMS (ESI) found m/z549.1643 (M+Na). C₃₀H₂₆N₂NaO₇ requires 549.1632.

A mixture of 66d (178 mg, crude, ca. 0.42 mmol), 66h (55 mg, 0.10 mmol),EDCI hydrochloride (160 mg, 0.84 mmol) and p-toluenesulfonic acid (1.8mg, 0.01 mmol) in DMA (2 mL) was stirred at room temperature overnight.The mixture was redistributed between ethyl acetate and cold diluteaqueous NaHCO₃. The aqueous phase was extracted with ethyl acetate threetimes. The combined organic extracts were washed with water followed bybrine, dried over anhydrous Na₂SO₄, and filtered through celite. Thesolvent was removed and the residue was further purified by Florisil®(US Silica) column chromatography using gradient mixtures of MeOH andethyl acetate (v/v 0.25-5%) to give (9H-fluoren-9-yl)methyl3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-(di-tert-butoxyphosphoryloxy)-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropylcarbamate66i as a yellow solid (62 mg, 44%); ¹H NMR (DMSO) δ 10.04 (s, 1H), 8.67(s, 2H), 8.11-8.05 (m, 3H), 7.97 (d, J=8.2 Hz, 2H), 7.88-7.82 (m, 4H),7.78 (d, J=8.4 Hz, 1H), 7.73-7.67 (m, 3H), 7.63-7.58 (m, 2H), 7.53-7.47(m, 3H), 7.38 (t, J=7.4 Hz, 2H), 7.30-7.24 (m, 4H), 4.60-4.50 (m, 4H),4.42-4.35 (m, 2H), 4.30 (d, J=6.7 Hz, 2H), 4.22 (t, J=6.6 Hz, 1H),4.06-3.93 (m, 4H), 3.40-3.33 (m, 2H), 2.63-2.59 (m, 2H), 1.50 (2×s,18H), 1.47 (2×s, 18H) ppm. ³¹P NMR (DMSO) δ −15.45 ppm. HRMS (ESI) foundm/z 1341.4677 (M+H). C₇₂H₈₁Cl₂N₄O₁₃P₂ requires 1341.4647.

To a solution of 66i (60 mg, 0.045 mmol) in DMF (1 mL) was addedpiperidine (44 μL, 0.45 mmol). The mixture was stirred at roomtemperature for 3 h then all the volatile components were pumped off.The resultant residue was triturated with a mixture of ether andpetroleum ether (v/v 1:1) to give free amine 66j as a white solid (44mg, 96%). ¹H NMR (DMSO) δ 8.68 (s, 2H), 8.11-8.05 (m, 3H), 7.98 (d,J=8.4 Hz, 2H), 7.91-7.86 (m, 2H), 7.78-7.85 (m, 1H), 7.70 (d, J=15.3 Hz,1H), 7.63-7.58 (m, 2H), 7.51 (t, J=7.9 Hz, 2H), 7.26 (d, J=15.3 Hz, 2H),4.66-4.52 (m, 4H), 4.44-4.35 (m, 2H), 4.07-3.95 (m, 4H), 2.95 (t, J=6.5Hz, 2H), 2.56-2.50 (m, 2H), 1.50-1.49 (m, 36H) ppm. ³¹P NMR (DMSO) δ−15.42, 15.45 ppm. HRMS (ESI) found m/z 1119.3981 (M+H).C₅₇H₇₁Cl₂N₄O₁₁P₂ requires 1119.3966.

A mixture of 66j (40 mg, 0.036 mmol), 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (SuOMC, 12 mg, 0.037mmol), and DIPEA (6.8 μL, 0.039 mmol) in DMSO (1 mL) was stirred at roomtemperature overnight before all the volatile components were pumpedoff. The resultant residue was triturated with ether to give bis(di-tert-butylphosphate)N-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide66k as a yellow solid (32 mg, 67%). ¹H NMR (DMSO) δ 10.02 (s, 1H), 8.67(s, 2H), 8.11-8.05 (m, 3H), 7.98-7.92 (m, 3H), 7.85-7.75 (m, 2H), 7.70(d, J=15.3 Hz, 1H), 7.63-7.58 (m, 2H), 7.51 (t, J=7.9 Hz, 2H), 7.26 (dd,J=4.4, 15.4 Hz, 2H), 6.96 (s, 2H), 4.66-4.52 (m, 4H), 4.44-4.35 (m, 2H),4.07-3.95 (m, 4H), 3.45-3.30 (m, 4H), 2.52-2.50 (m, 2H), 2.08 (t, J=7.2Hz, 2H), 1.50-1.40 (m, 40H), 1.33-1.25 (m, 2H) ppm. ³¹P NMR (DMSO) δ−15.42, 15.45 ppm. HRMS (ESI) found m/z 1334.4515 (M+Na).C₆₇H₈₁Cl₂N₅NaO₁₄P₂ requires 1334.4525.

To a solution of 66k (30 mg, 0.02 mmol) in DCM (1 mL) cooled in an icebath was added TFA (1 mL, 12.98 mmol). The mixture was allowed to warmup to room temperature and stirred for 3 h. All the volatile componentswere pumped off and the resultant residue was triturated with ethylacetate to giveN-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-phosphonoxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide66 as a yellow solid (20 mg, 80%, HPLC purity 95%); ¹H NMR (DMSO) δ10.01 (s, 1H), 8.59 (s, 2H), 8.16-8.08 (m, 3H), 7.97-7.90 (m, 3H),7.88-7.73 (m, 2H), 7.69 (d, J=14.5 Hz, 1H), 7.61-7.55 (m, 2H), 7.46 (t,J=7.3 Hz, 2H), 7.29-7.24 (d, J=14.0 Hz, 2H), 6.97 (s, 2H), 4.62-4.52 (m,3H), 4.40-4.30 (m, 3H), 4.05-3.90 (m, 4H), 3.43-3.37 (m, 2H), 3.24-3.18(m, 2H), 2.61-2.55 (m, 2H), 2.08 (br s, 2H), 1.54-1.44 (m, 4H),1.27-1.15 (m, 2H) ppm. ³¹P NMR (DMSO) δ −5.81 ppm. HRMS (ESI negative)found m/z 1086.2067 (M−H). C₅₁H₄₈Cl₂N₅O₁₄P₂ requires 1086.2056.

Example 17N-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide67

To a solution of (2E,2′E)-tert-butyl3,3′-(2-(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)propanamido)-1,4-phenylene)diacrylate66g (4.89 g, 7.66 mmol) in DCM (30 mL) was added piperidine (4.5 mL,45.50 mmol). See FIG. 23. The mixture was stirred at room temperaturefor 5 h before all the volatile components were removed by rotaryevaporator. Column chromatography using ethyl acetate as eluent followedby a mixture of TEA, MeOH, and ethyl acetate (v/v 1:10:100) gave(2E,2′E)-tert-butyl3,3′-(2-(3-aminopropanamido)-1,4-phenylene)diacrylate 67a as a whitesolid (2.33 g, 73%). mp 62-63° C. ¹H NMR (CDCl₃) δ 11.04 (s, 1H), 8.32(d, J=1.4 Hz, 1H), 7.91 (d, J=15.7 Hz, 1H), 7.55 (d, J=16.1 Hz, 1H),7.54 (d, J=8.0 Hz, 1H), 7.24 (dd, J=1.5, 8.2 Hz, 1H), 6.42 (d, J=16.0Hz, 1H), 6.34 (d, J=15.7 Hz, 1H), 3.18 (d, J=5.6 Hz, 2H), 2.52 (t, J=5.6Hz, 2H), 1.53 (s, 18H) ppm. HRMS (ESI) found m/z 417.2394 (M+H).C₂₃H₃₃N₂O₅ requires 417.2384.

A mixture of 67a (1.00 g, 2.40 mmol), 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (SuOMC, 740 mg, 2.40mmol), and DIPEA (460 μL, 2.64 mmol) in DMF (10 mL) was stirred at roomtemperature overnight. All the volatile components were pumped off. Theresultant residue was purified by column chromatography using a mixtureof ethyl acetate and petroleum ether (v/v 2:1) as eluent, followed byethyl acetate alone, to give (2E,2′E)-tert-butyl3,3′-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-1,4-phenylene)diacrylate67b as a white solid (1.00 g, 68%). mp 69-72° C. ¹H NMR (CDCl₃) δ 7.95(s, 1H), 7.73 (s, 1H), 7.65 (d, J=15.9 Hz, 1H), 7.61 (d, J=8.2 Hz, 1H),7.53 (d, J=16.0 Hz, 1H), 7.37 (d, J=8.2 Hz, 1H), 6.69 (br s, 1H), 6.64(s, 2H), 6.39 (d, J=16.0 Hz, 1H), 6.38 (d, J=15.8 Hz, 1H), 3.68-3.63 (m,2H), 3.43 (d, J=7.0 Hz, 2H), 2.69 (d, J=5.5 Hz, 2H), 2.21 (t, J=7.3 Hz,2H), 1.67-1.50 (m, 4H), 1.53 (s, 9H), 1.52 (s, 9H), 1.31-1.24 (m, 2H)ppm. HRMS (ESI) found m/z 632.2931 (M+Na). C₃₃H₄₃N₃NaO₈ requires632.2942.

To a solution of 67b (500 mg, 0.82 mmol) in DCM (4 mL) cooled in an icebath was added TFA (2 mL) dropwise. The mixture was allowed to warm upto room temperature and stirred for 4 h. After all volatile componentswere pumped off, the resultant residue was triturated with ethyl acetateto give(2E,2′E)-3,3′-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-1,4-phenylene)diacrylicacid 67c as a white solid (380 mg, 93%). mp 257-261° C. ¹H NMR (DMSO) δ12.47 (s, 2H), 9.90 (s, 1H), 7.89-7.83 (m, 2H), 7.71-7.67 (m, 2H),7.57-7.53 (m, 2H), 6.99 (s, 2H), 6.53 (d, J=15.9 Hz, 2H), 3.36-3.30 (m,4H), 2.54-2.50 (m, 2H), 2.06 (t, J=7.3 Hz, 2H), 1.52-1.42 (m, 4H),1.22-1.17 (m, 2H) ppm. HRMS (ESI) found m/z 520.1683 (M+Na).C₂₅H₂₇N₃NaO₈ requires 520.1690.

To a solution of (S)-tert-Butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a (298mg, 0.89 mmol) in DCM (5 mL) cooled in an ice bath was added 4N HCl indioxane (10 mL). The mixture was allowed to warm up to room temperatureand stirred for 3 h. All volatile components were pumped off to give(S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol 67d as ahydrochloride salt to which was added 67c (185 mg, 0.37 mmol), EDCIhydrochloride (428 mg, 2.23 mmol), p-toluenesulfonic acid (6 mg, 0.037mmol) and DMA (5 mL). After the mixture was stirred at room temperaturefor 6 h, more EDCI hydrochloride (285 mg, 1.49 mmol) and toluenesulfonicacid (6 mg, 0.037 mmol) were added. The mixture was stirred overnightand then redistributed between ethyl acetate and water. The aqueousphase was extracted with ethyl acetate three times. The combined organicextracts were washed with water followed by brine, dried over anhydrousNa₂SO₄, and filtered through celite. The solvent was removed and theresultant residue was further purified by column chromatography usinggradient mixtures of MeOH and ethyl acetate (v/v 1-15%) as eluent togive 67 as a yellow solid (160 mg, 46%, HPLC purity 96%); mp 230-234° C.(dec). ¹H NMR (DMSO) δ 10.43 (s, 1H), 10.40 (s, 1H), 10.00 (s, 1H),8.14-8.08 (m, 5H), 7.95 (br s, 1H), 7.83-7.75 (m, 5H), 7.68 (d, J=15.2Hz, 1H), 7.52 (t, J=7.4 Hz, 2H), 7.35 (t, J=7.6 Hz, 2H), 7.28-7.23 (dd,J=5.2, 15.2 Hz, 2H), 6.95 (s, 2H), 4.58-4.45 (m, 4H), 4.29-4.20 (m, 2H),4.04-3.97 (m, 2H), 3.88-3.80 (m, 2H), 3.43-3.37 (m, 2H), 3.23 (d, J=6.9Hz, 2H), 2.60-2.56 (m, 2H), 2.09 (d, J=7.1 Hz, 2H), 1.50-1.40 (m, 2H),1.40-1.30 (m, 2H), 1.24-1.14 (m, 2H) ppm. HRMS (ESI) found m/z 950.2672(M+Na). C₅₁H₄₇Cl₂N₅NaO₈ requires 950.2694.

Example 18(S)-1-(chloromethyl)-3-((E)-3-(4-((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)-2-(3-(6-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)hexanamido)propanamido)phenyl)acryloyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 68

To a solution of (S)-di-tert-butyl1-(chloromethyl)-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-5-ylphosphate 66c (125 mg, 0.24 mmol) in MeOH (1 mL) cooled in an ice bathwas added CsCO₃ (298 mg, 0.92 mmol) and several drops of water. Themixture was stirred in an ice bath for 1 h and then redistributedbetween ethyl acetate and water. The aqueous phase was extracted withethyl acetate three times. The combined organic extracts were washedwith water and brine, dried over anhydrous Na₂SO₄, filtered throughcelite, and the solvent was removed. The resultant residue was dissolvedin ethyl acetate and filtered through a pad of Florisil® (US Silica).Solvent removal gave (S)-di-tert-butyl1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl phosphate 66d as anoff-white gum, which was used directly without further purification.

To a solution of (S)-tert-Butyl1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate 51a (80mg, 0.24 mmol) in DCM (2 mL) cooled in an ice bath was added of 4N HClin dioxane (4 mL). The mixture was allowed to warm up to roomtemperature and stirred for 2 h. All volatile components were pumped offand (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol 67d was useddirectly.

To a solution of 66d and 67d (each prepared as above) in DMA (2 mL)cooled in a salt-ice bath (−10° C.),(2E,2′E)-3,3′-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-1,4-phenylene)diacrylicacid 67c (119 mg, 0.24 mmol) was added followed by EDCI hydrochloride(276 mg, 1.44 mmol) and p-toluenesulfonic acid (4 mg, 0.024 mmol). Themixture was allowed to warm up to room temperature, stirred overnight,and then poured on ice. The resultant precipitate was collected byfiltration, washed with water, and dried under vacuum. Purification byFlorisil® (US Silica) chromatography column using gradient mixtures ofMeOH and DCM (v/v 2-10%) gave a yellow solid (50 mg), which wasidentified by LC-MS as a mixture of di-tert-butyl((S)-1-(chloromethyl)-3-((E)-3-(4-((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-en-1-yl)-2-(3-(6-(2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)phenyl)acryloyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)phosphate 68a and di-tert-butyl((S)-1-(chloromethyl)-3-((E)-3-(4-((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-en-1-yl)-3-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)phenyl)acryloyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)phosphate 68b (20% and 23% respectively by HPLC), 67 (44% by HPLC), and66 (8% by HPLC). The mixture was further purified by preparative HPLC(Column: Synergi-Max® RP 4μ, 250×21.20 mm; Mobile phase: A/B=from 25% to0% (A: ammonium formate pH 3.45, B: 90% acetonitrile in water); flowrate 12 mL/min, gradient method; wave length: 254 nm, 325 nm) to give amixture of 68a and 68b as a yellow solid (18 mg, 8%). HRMS (ESI) foundm/z 1142.3577 (M+Na). C₅₉H₆₄Cl₂N₅NaO₁₁P requires 1142.3609.

To a solution of 68a and 68b (17 mg, 0.015 mmol) in DCM (0.5 mL) cooledin an ice bath was added TFA (0.5 mL) dropwise. The mixture was allowedto warm up to room temperature and stirred for 3 h. All volatilecomponents were pumped off. The resultant residue was triturated withethyl acetate to give a mixture of(S)-1-(chloromethyl)-3-((E)-3-(4-((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)-2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)phenyl)acryloyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 68 and phosphate regioisomer,(S)-1-(chloromethyl)-3-((E)-3-(4-((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)-3-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)phenyl)acryloyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate 68c as an orange solid (11 mg, 72%, HPLC purity:95%, ratio of isomers 1:1). ¹H NMR (DMSO) δ 10.45 (s, 1H), 10.01 (s,1H), 8.58 (s, 1H), 8.15-7.23 (m, 17H), 6.95 (s, 2H), 4.60-3.80 (m, 10H),3.43-3.37 (m, 2H), 3.27-3.18 (m, 2H), 2.60-2.50 (m, 2H), 2.10-2.00 (m,2H), 1.55-1.35 (m, 4H), 1.20-1.10 (m, 2H) ppm. ³¹P NMR (DMSO) δ −5.81ppm. HRMS (ESI negative) found m/z 1006.2394 (M−H). C₅₁H₄₇Cl₂N₅O₁₁Prequires 1006.2392.

Example 23N-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide(Compound No. 69, Table 4, FIG. 37)

To 192 mg (0.53 mmol) of 1 (freshly made by the procedure mentionedabove) in DMA (2 mL) was added 2 (100 mg, 0.20 mmol), EDCI hydrochloride(231 mg, 1.21 mmol) and toluenesulfonic acid (3.5 mg, 0.020 mmol). Themixture was stirred overnight and then poured into a mixture of aqueousammonia and ice. The resulting precipitate was collected by filtration,washed with water, dried and purified by silica gel columnchromatography. Gradient mixtures of MeOH and DCM (v/v 1-15%) were usedas eluent to provideN-(3-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyloxy)-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamideas a yellow solid (30 mg, 13%, HPLC purity 96%). mp 268° C. (dec). ¹HNMR (CDCl₃) δ 8.60 (s, 1H), 8.46 (s, 1H), 8.42 (s, 1H), 7.87-7.84 (m,3H), 7.75-7.60 (m, 5H), 7.40-7.33 (m, 5H), 7.22 (br s, 1H), 6.77 (t,J=15.3 Hz, 2H), 6.56 (s, 2H), 4.46-4.40 (m, 2H), 4.30-4.25 (m, 2H),4.11-4.00 (m, 2H), 3.95-3.90 (m, 7H), 3.68 (apparent s, 6H), 3.53-3.46(m, 3H), 3.38 (t, J=7.0 Hz, 2H), 2.73 (apparent s, 2H), 2.59-2.55 (m,8H), 2.42 (s, 6H), 2.28 (t, J=7.0 Hz, 2H), 1.56-1.49 (m, 2H), 1.31-1.23(m, 2H) ppm. HRMS (ESI) found m/z 1180.4416 (M+H). C₆₃H₆₈Cl₂N₉O₁₀requires 1180.4416.

Example 24 2-(pyridin-2-yldisulfanyl)propyl2,5-bis((E)-3-((S)-1-(chloromethyl)-5-(phosphonooxy)-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenylcarbamate(Table 4, Compound No. 72, FIG. 38)

To 207 mg (0.49 mmol) of 1 (freshly made by the procedure mentionedabove) was added 2 (70 mg, 0.15 mmol), EDCI hydrochloride (233 mg, 1.22mmol), toluenesulfonic acid (3 mg, 0.015 mmol) and DMA (0.5 mL). Afterthe mixture was stirred overnight, most of the DMA was removed undervacuum and the residue was redistributed between ethyl acetate andwater. The aqueous phase was extracted with ethyl acetate three times.The combined organic extracts were washed with water followed by brine,dried over anhydrous Na₂SO₄, and filtered through a pad of Celite. Thesolvent was removed and the resultant residue was dissolved in theminimum DCM and precipitated by adding heptane to give crude product(156 mg), which was further purified by preparative HPLC (Column:Synergi-Max RP 4μ, 250×21.20 mm; Mobile phase: A/B=from 10% to 1% (A:ammonium formate pH 3.45, B: 90% acetonitrile in water); flow rate 12mL/min, gradient method; wavelength: 254 nm, 325 nm) to give 3 (78 mg,40%) as a yellow solid. ¹H NMR (DMSO) δ 9.67 (s, 1H), 8.67 (s, 2H), 8.43(d, J=4.5 Hz, 1H), 8.11-8.06 (m, 3H), 7.97 (apparent d, J=8.5 Hz, 2H),7.92 (d, J=15.3 Hz, 1H), 7.84-7.76 (m, 3H), 7.70 (d, J=15.3 Hz, 2H),7.61 (t, J=7.6 Hz, 2H), 7.51 (t, J=7.7 Hz, 2H), 7.28 (d, J=15.3 Hz, 1H),7.27 (d, J=15.3 Hz, 1H), 7.23-7.19 (m, 1H), 4.62-4.53 (m, 4H), 4.43-4.37(m, 2H), 4.23-4.13 (m, 2H), 4.05-3.95 (m, 4H), 3.42-3.37 (m, 1H), 1.51(s, 9H), 1.50 (s, 9H), 1.49 (s, 9H), 1.48 (s, 9H), 1.34 (d, J=6.5 Hz,3H) ppm. ³¹P NMR (DMSO) δ −15.46 (s) and −15.48 (s) ppm. HRMS (ESI)found m/z 1297.3471 (M+Na). C₆₃H₇₄Cl₂N₄NaO₁₂P₂S₂ requires 1297.3489.

To a solution of 3 (60 mg, 0.047 mmol) in DCM (2 mL) cooled in an icebath was added TFA (1 mL, 6.49 mmol). The mixture was allowed to warm upto room temperature and stirred for 3 h. All the volatile componentswere pumped off and the resultant residue was triturated with ethylacetate to give 4 as a yellow solid (49 mg, 99%, HPLC purity 100%); ¹HNMR (DMSO) δ 9.65 (s, 1H), 8.59 (s, 2H), 8.46-8.44 (m, 1H), 8.15-8.08(m, 3H), 7.96-7.90 (m, 3H), 7.82-7.70 (m, 3H), 7.70 (d, J=15.6 Hz, 1H),7.59 (t, J=6.8 Hz, 2H), 7.48 (t, J=7.5 Hz, 2H), 7.31-7.22 (m, 3H), 5.75(s, 1H), 4.64-4.53 (m, 4H), 4.40-4.33 (m, 3H), 4.25-4.15 (m, 3H),4.06-3.93 (m, 3H), 1.35-1.32 (m, 3H) ppm. ³¹P NMR (DMSO) δ −5.95 (s)ppm. HRMS (ESI) found m/z 1073.0949 (M+Na). C₄₇H₄₂Cl₂N₄NaO₁₂P₂S₂requires 1073.0985.

Example 25[(1S)-1-(chloromethyl)-3-[(E)-3-[4-[(E)-3-[(1S)-1-(chloromethyl)-5-phosphonooxy-1,2-dihydrobenzo[e]indol-3-yl]-3-oxo-prop-1-enyl]-2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]phenyl]prop-2-enoyl]-1,2-dihydrobenzo[e]indol-5-yl]dihydrogen phosphate (Compound No. 78, Table 4, FIG. 39)

Intermediate 3

To 76 mg (0.18 mmol) of 1 (freshly made by the procedure mention above)was added 2 (18 mg, 0.045 mmol), EDCI hydrochloride (69 mg, 0.36 mmol),toluenesulfonic acid (0.8 mg, 0.005 mmol) and DMA (0.25 mL). After themixture was stirred overnight, most of DMA was removed under vacuum andthe residue was redistributed between ethyl acetate and water. Theaqueous phase was extracted with ethyl acetate three times. The combinedorganic extracts were washed with water followed by brine, dried overanhydrous Na₂SO₄, and filtered through a pad of Celite. The solvent wasremoved and the resultant residue was dissolved in the minimum DCM andprecipitated by adding heptane to give crude product (54 mg), which wasfurther purified by preparative HPLC (Column: Synergi-Max RP 4μ,250×21.20 mm; Mobile phase: A/B=from 20% to 1% (A: ammonium formate pH3.45, B: 90% acetonitrile in water); flow rate 12 mL/min, gradientmethod; wavelength: 254 nm, 325 nm) to give 3 (17 mg, 30%) as a yellowsolid. ¹H NMR (CDCl₃) δ 8.72 (br s, 2H), 8.23 (d, J=8.4 Hz, 2H), 7.96(d, J=15.2 Hz, 1H), 7.83 (d, J=15.3 Hz, 1H), 7.71 (d, J=8.2 Hz, 2H),7.54-7.50 (m, 3H), 7.42-7.39 (m, 2H), 7.26-7.12 (m, 3H), 6.95-6.88 (m,1H), 6.67 (s, 2H), 4.57-4.52 (m, 2H), 4.47-4.38 (m, 2H), 4.28-4.24 (m,2H), 4.16-4.09 (m, 2H), 4.00-3.94 (m, 4H), 3.78 (apparent s, 4H),3.55-0.348 (m, 2H), 1.57 (s, 36H) ppm. ³¹P NMR (CDCl₃) δ −15.64 (s) ppm.HRMS (ESI) found m/z 1238.3862 (M+Na). C₆₂H₇₃Cl₂N₃NaO₁₄P₂ requires1238.3837.

To a solution of 3 (16 mg, 0.013 mmol) in DCM (1 mL) cooled in an icebath was added TFA (0.5 mL, 3.24 mmol). The mixture was allowed to warmup to room temperature and stirred for 3 h. All the volatile componentswere pumped off and the resultant residue was triturated with ethylacetate to give Compound 78 as a yellow solid (13 mg, 100%, HPLC purity100%). ¹H NMR (DMSO) δ 8.60 (s, 2H), 8.12 (d, J=8.4 Hz, 2H), 7.95-7.87(m, 4H), 7.72 (d, J=15.1 Hz, 1H), 7.61-7.57 (m, 2H), 7.53-7.45 (m, 4H),7.38-7.32 (m, 2H), 6.97 (s, 2H), 4.60-4.48 (m, 4H), 4.30-4.28 (m, 4H),4.08-3.88 (m, 6H), 3.68-3.58 (m, 4H). ³¹P NMR (DMSO) δ −5.94 (s) ppm.HRMS (ESI) found m/z 1014.1301 (M+Na). C₄₆H₄₁Cl₂N₃NaO₁₄P₂ requires1014.1333.

Example 26[(1S)-1-(chloromethyl)-3-[(E)-3-[4-[(E)-3-(1S)-1-(chloromethyl)-5-phosphonooxy-1,2-dihydrobenzo[e]indol-3-yl-3-oxo-prop-1-enyl]-2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]phenyl]prop-2-enoyl]-1,2-dihydrobenzo[e]indol-5-yl]dihydrogen phosphate (Compound No. 79, Table 4, FIG. 40)

Intermediate 3

To 52 mg (0.12 mmol) of 1 (freshly made by the procedure mentionedabove) was added 2 (11 mg, 0.031 mmol), EDCI hydrochloride (35 mg, 0.18mmol), toluenesulfonic acid (0.5 mg, 0.003 mmol) and DMA (0.25 mL).After the mixture was stirred overnight, most of the DMA was removedunder vacuum and the residue was redistributed between ethyl acetate andwater. The aqueous phase was extracted with ethyl acetate three times.The combined organic extracts were washed with water followed by brine,dried over anhydrous Na₂SO₄, and filtered through a pad of Celite. Thesolvent was removed and the resultant residue was dissolved in theminimum DCM and precipitated by adding heptane to give crude product (71mg), which was further purified by preparative HPLC (Column: Synergi-MaxRP 4μ, 250×21.20 mm; Mobile phase: A/B=from 20% to 1% (A: ammoniumformate pH 3.45, B: 90% acetonitrile in water); flow rate 12 mL/min,gradient method; wavelength: 254 nm, 300 nm) to give 3 (15 mg, 42%) as ayellow solid. ¹H NMR (CDCl₃) δ 8.71 (br s, 2H), 8.25 (d, J=8.4 Hz, 2H),7.95 (d, J=15.5 Hz, 1H), 7.83 (d, J=15.3 Hz, 1H), 7.72 (d, J=8.2 Hz,2H), 7.60-7.52 (m, 3H), 7.46-7.40 (m, 2H), 7.28-7.20 (m, 2H), 7.13-6.99(m, 2H), 6.78 (s, 2H), 4.64-4.60 (m, 2H), 4.51-4.45 (m, 2H), 4.38-4.32(m, 2H), 4.15-4.05 (m, 4H), 4.00-3.95 (m, 2H), 3.57-3.49 (m, 2H), 1.58(s, 36H) ppm. ³¹P NMR (CDCl₃) δ −15.67 (s) ppm. HRMS (ESI) found m/z1194.3606 (M+Na). C₆₀H₆₉Cl₂N₃NaO₁₃P₂ requires 1194.3575.

Compound No. 79

To a solution of 3 (13 mg, 0.011 mmol) in DCM (0.5 mL) cooled in an icebath was added TFA (0.2 mL, 1.30 mmol). The mixture was allowed to warmup to room temperature and stirred for 0.5 h. Diethyl ether was added togive a precipitate, which was filtered off and washed with ethyl acetateto give 4 (Compound No. 79) as a yellow solid (8.4 mg, 80%, HPLC purity90%). ¹H NMR (DMSO) δ 8.59 (s, 2H), 8.13 (d, J=8.3 Hz, 2H), 7.97-7.82(m, 4H), 7.71 (d, J=15.1 Hz, 1H), 7.60-7.52 (m, 3H), 7.49-7.44 (m, 3H),7.35-7.24 (m, 2H), 7.08 (s, 2H), 4.65-4.50 (m, 4H), 4.40-4.30 (m, 4H),4.05-3.90 (m, 6H). ³¹P NMR (DMSO) δ −5.81 (s) ppm. HRMS (ESI) found m/z970.1036 (M+Na). C₄₄H₃₇Cl₂N₃NaO₃P₂ requires 970.1071.

Example 27 2-(2-pyridyldisulfanyl)propylN-[1-(chloromethyl)-3-[5-[1-(chloromethyl)-5-hydroxy-1,2-dihydrobenzo[e]indol-3-yl]-5-oxo-pentanoy]-1,2-dihydrobenzo[e]indol-5-yl]carbamate(Compound No. 80, Table 4, FIG. 41)

A mixture of 62c (31.0 mg, 0.0674 mmol, freshly made by the procedurementioned above), 53h (23.0 mg, 0.0674 mmol, freshly made by theprocedure mentioned above), EDCI.HCl (38.7 mg, 0.202 mmol) and TsOH (2.3mg, 0.0135 mmol) in dry DMA (2 mL) was stirred at r.t. overnight, undernitrogen. After 18 h the reaction mixture was diluted with EtOAc and H₂Oand well mixed. The layers were separated and the organic layer washedwith H₂O (3×), dried (Na₂SO₄) and solvent removed under vacuum. Thecrude product was purified by column chromatography on silica gel usingDCM:MeOH 100:0 to 98:2, followed by trituration with diisopropyl etherto give compound 1 (Compound No. 80, 26.0 mg, 49%, HPLC purity: 95.2%)as a cream solid. ¹H NMR δ (400 MHz, DMSO-d₆) 10.36 (s, 1H), 9.69 (s,1H), 8.56 (s, 1H), 8.44 (d, J=4.3 Hz, 1H), 8.08 (d, J=8.0 Hz, 1H),8.02-8.00 (m, 2H), 7.92 (d, J=8.3 Hz, 1H), 7.85-7.77 (m, 3H), 7.57-7.53(m, 1H), 7.51-7.47 (m, 1H), 7.45-7.41 (m, 1H), 7.34-7.30 (m, 1H),7.24-7.21 (m, 1H), 4.42-4.29 (m, 3H), 4.25-4.13 (m, 5H), 4.05-3.97 (m,2H), 3.91 (dd, J=11, 7.2 Hz, 1H), 3.79 (dd, J=11, 8.3 Hz, 1H), 3.43-3.36(m, 1H), 2.77-2.56 (m, 4H), 2.01-1.93 (m, 2H), 1.34 (d, J=6.7 Hz, 3H).HRMS m/z 811.1542 [(M+Na)⁺ calcd for C₄₀H₃₈Cl₂N₄NaO₅S₂ 811.1553].

Examples 28 2-(2-pyridyldisulfanyl)propyl3-[6-[1-(chloromethyl)-5-(4-methylpiperazine-1-carbonyl)oxy-1,2-dihydrobenzo[e]indol-3-yl]-6-oxo-hexoxy]-6-hydroxy-2-methoxy-11-oxo-6a,7,8,9-tetrahydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate,2-(2-pyridyldisulfanyl)propyl3-[6-[1-(chloromethyl)-5-phosphonooxy-1,2-dihydrobenzo[e]indol-3-yl]-6-oxo-hexoxy]-6-hydroxy-2-methoxy-11-oxo-6a,7,8,9-tetrahydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate,and 2-(2-pyridyldisulfanyl)propyl3-[6-[1-(chloromethyl)-5-hydroxy-1,2-dihydrobenzo[e]indol-3-yl]-6-oxo-hexoxy]-6-hydroxy-2-methoxy-11-oxo-6a,7,8,9-tetrahydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate(Compound No. 81-83, Table 4, FIG. 42-43)

A mixture of 1 (1.40 g, 4.00 mmol, prepared following literatureprocedure: J. Med. Chem. 2003, 46, 2132-2151), 2 (1.31 g, 5.22 mmol,prepared following literature procedure: WO2004065491 A1) and K₂CO₃ (829mg, 6.00 mmol) in dry DMA (15 mL) was stirred at r.t. for 43 h. Themixture was then diluted with EtOAc and H₂O, well mixed and the layersseparated. The organic layer was washed with H₂O (3×), brine (1×) anddried (Na₂SO₄) and the solvent removed under vacuum. The crude productwas purified by column chromatography on silica gel using EtOAc:Hex50:50 to 67:33 to 100:0 to give compound 3 (1.74 g, 84%) as a yellowoil. ¹H NMR δ (400 MHz, CDCl₃) 8.77 (br s, 1H), 7.79 (s, 1H), 6.82 (s,1H), 6.02-5.92 (m, 1H), 5.36 (dq, J=17.2, 1.5 Hz, 1H), 5.26 (dq, J=10.4,1.2 Hz, 1H), 4.69-4.60 (m, 2H), 4.47-4.39 (m, 1H), 4.28 (br s, 1H),4.09-4.05 (m, 2H), 3.83 (s, 3H), 3.90-3.80 (m, 1H), 3.74-3.70 (m, 1H),3.65-3.59 (m, 1H), 3.54-3.47 (m, 1H), 2.25 (t, J=7.4 Hz, 2H), 2.20-2.15(m, 1H), 1.93-1.84 (m, 3H), 1.81-1.72 (m, 1H), 1.70-1.63 (m, 3H),1.53-1.47 (m, 2H), 1.44 (s, 9H). HRMS m/z 543.2666 [(M+Na)⁺ calcd forC₂₇H₄₀N₂NaO₈ 543.2677].

Et₃N (1.32 mL, 9.47 mmol) was added to a solution of 3 (821 mg, 1.58mmol) in dry DCM (6 mL) at r.t. Acetic anhydride (0.75 mL, 7.93 mmol)was then added and the mixture stirred at r.t. for 4.5 h. The reactionmixture was cooled to 0° C. and dry MeOH (1 mL) added and the mixturestirred at 0° C. for 15 mins. EtOAc (120 mL) was then added and themixture washed with H₂O (2×), brine (1×), dried (Na₂SO₄) and solventremoved under vacuum to give compound 4 (891 mg, quantitative) which wasused in the next step without purification. ¹H NMR δ (400 MHz, CDCl₃)8.88 (br s, 1H), 7.82 (s, 1H), 6.81 (s, 1H), 6.01-5.91 (m, 1H), 5.36(dq, J=17.2, 1.5 Hz, 1H), 5.25 (dq, J=10.4, 1.3 Hz, 1H), 4.65-4.62 (m,2H), 4.61-4.54 (m, 1H), 4.32-4.22 (m, 2H), 4.09-4.06 (m, 2H), 3.83 (s,3H), 3.55-3.47 (m, 2H), 2.26-2.23 (m, 2H), 2.18-2.12 (m, 1H), 2.07 (s,3H), 1.97-1.77 (m, 5H), 1.70-1.63 (m, 2H), 1.54-1.47 (m, 2H), 1.44 (s,9H). HRMS m/z 585.2774 [(M+Na)⁺ calcd for C₂₉H₄₂N₂NaO₉ 585.2783].

Pyrrolidine (1.6 mL, 19.2 mmol) was added to a solution of 4 (1.06 g,1.88 mmol) in dry DCM (20 mL) at r.t. Pd(PPh₃)₄(109 mg, 0.0943 mmol) wasthen added and the reaction mixture stirred at r.t. for 40 mins. Thereaction mixture was washed with 0.25 M HCl solution (2×75 mL), dried(Na₂SO₄) and solvent removed under vacuum. The crude product waspurified by column chromatography on silica gel using EtOAc:Hex 50:50 to100:0 to give compound 5 (726 mg, 81%) as a yellow oil. ¹H NMR δ (400MHz, DMSO-d₆) 6.67 (s, 1H), 6.35 (s, 1H), 5.08 (s, 2H), 4.35-4.30 (m,1H), 4.13-4.06 (m, 2H), 3.87 (t, J=6.4 Hz, 2H), 3.63 (s, 3H), 3.50-3.44(m, 1H), 3.42-3.35 (m, 1H), 2.21 (t, J=7.2 Hz, 2H), 2.07-2.00 (m, 1H),2.01 (s, 3H), 1.89-1.82 (m, 1H), 1.77-1.67 (m, 4H), 1.59-1.51 (m, 2H),1.44-1.36 (m, 2H), 1.39 (s, 9H). HRMS m/z 501.2573 [(M+Na)⁺ calcd forC₂₅H₃₈N₂NaO₇ 501.2571].

Diphosgene (0.22 mL, 1.82 mmol) was added to a mixture of 5 (726 mg,1.52 mmol) and DMAP (557 mg, 4.56 mmol) in dry DCM (25 mL) at r.t. undernitrogen. After 30 mins a solution of 6 (2.60 g, 12.9 mmol; freshly madeby the procedure mentioned above—no number previously assigned toalcohol) in dry DCM (25 mL) was added and the mixture stirred at r.t.overnight. After 18 h the reaction mixture was washed with H₂O (1×),dried (Na₂SO₄) and solvent removed under vacuum. The crude product waspurified by column chromatography on silica gel using DCM:EtOAc 100:0 to95:5 to 94:6 until excess 6 eluted and then EtOAc:Hex 70:30 to givecompound 7 (920 mg, 86%) as a pale yellow oil. ¹H NMR δ (400 MHz,DMSO-d₆) 9.16 (br s, 1H), 8.45-8.43 (m, 1H), 7.83-7.78 (m, 2H),7.25-7.21 (m, 1H), 7.15 (d, J=2.8 Hz, 1H), 6.87 (s, 1H), 4.29 (br s,1H), 4.17-3.99 (m, 4H), 3.92 (t, J=6.4 Hz, 2H), 3.75 (s, 3H), 3.42-3.30(m, 3H), 2.20 (t, J=7.2 Hz, 2H), 2.06-1.95 (m, 4H), 1.83 (br s, 1H),1.77-1.68 (m, 4H), 1.58-1.49 (m, 2H), 1.43-1.36 (m, 2H), 1.39 (s, 9H),1.29 (d, J=6.8 Hz, 3H). HRMS m/z 706.2832 [(M+H)⁺ calcd for C₃₄H₄₈N₃O₉S₂706.2826].

A mixture of 7 (949 mg, 1.34 mmol) and K₂CO₃ (1.85 g, 13.4 mmol) inDCM-MeOH (34 mL/17 mL) was stirred at r.t. for 45 mins. The mixture wasdiluted with DCM, poured into ice H₂O (200 mL), well mixed and thelayers separated. The aqueous layer was extracted with DCM (1×), thecombined organic layers were dried (Na₂SO₄) and solvent removed undervacuum. The crude product was purified by column chromatography onsilica gel using DCM:EtOAc 100:0 to 50:50 to give compound 8 (808 mg,91%) as a pale yellow oil. ¹H NMR δ (400 MHz, DMSO-d₆) 9.20 (br s, 1H),8.44 (d, J=4.7 Hz, 1H), 7.81-7.80 (m, 2H), 7.25-7.20 (m, 2H), 6.94 (s,1H), 4.75 (t, J=5.6 Hz, 1H), 4.17-3.99 (m, 3H), 3.92 (t, J=6.4 Hz, 2H),3.74 (s, 3H), 3.60-3.46 (m, 2H), 3.37-3.20 (m, 3H), 2.20 (t, J=7.2 Hz,2H), 1.93-1.76 (m, 3H), 1.75-1.68 (m, 3H), 1.58-1.51 (m, 2H), 1.44-1.36(m, 2H), 1.39 (s, 9H), 1.29 (d, J=6.9 Hz, 3H). HRMS m/z 664.2721 [(M+H)⁺calcd for C₃₂H₄₆N₃O₈S₂ 664.2724].

(Diacetoxyiodo)benzene (259 mg, 0.804 mmol) was added to a mixture of 8(349 mg, 0.526 mmol) and TEMPO (82.2 mg, 0.526 mmol) in dry DCM (10 mL)at r.t. and the reaction mixture stirred overnight. After 24 h themixture was diluted with DCM and saturated aqueous Na₂S₂O₃ and wellmixed. The layers were separated and the organic layer was washed withsaturated aqueous Na₂S₂O₃ (1×), saturated aqueous NaHCO₃ (1×), dried(Na₂SO₄) and solvent removed under vacuum. The crude product waspurified by column chromatography on silica gel using EtOAc:Hex 70:30 to100:0 to give compound 9 (248 mg, 71%) as a white foam. ¹H NMR δ (400MHz, DMSO-d₆) 8.45-8.43 (m, 1H), 7.79-7.69 (m, 1H), 7.51-7.48 (m, 1H),7.24-7.20 (m, 1H), 7.10 (s, 1H), 6.96 and 6.91 (2s, 1H), 6.55 (t, J=5.9Hz, 1H), 5.46 (dd, J=8.9, 6.1 Hz, 1H), 4.31-4.21 (m, 1H), 4.02-3.84 (m,3H), 3.80 and 3.79 (2s, 3H), 3.52-3.46 (m, 1H), 3.40-3.18 (m, 3H),2.19-2.13 (m, 2H), 2.09-2.00 (m, 1H), 1.96-1.85 (m, 3H), 1.70-1.67 (m,2H), 1.56-1.45 (m, 2H), 1.40-1.34 (m, 2H), 1.38 and 1.37 (2s, 9H),1.15-1.10 (m, 3H). HRMS m/z 662.2592 [(M+H)⁺ calcd for C₃₂H₄₄N₃O₈S₂662.2564].

A mixture of 9 (254 mg, 0.384 mmol) and 4 M HCl in dioxane (11 mL) wasstirred at r.t. for 1 h 15 mins. The solvent was removed under vacuum at25-30° C. to give compound 10 (162 mg, 70%) which was used in the nextstep without purification.

A mixture of 10 (161 mg, 0.266 mmol), 58b (195 mg, 0.542 mmol, freshlymade by the procedure mentioned above), EDCI.HCl (253 mg, 1.32 mmol) andTsOH (19.5 mg, 0.113 mmol) in dry DMA (5 mL) was stirred at r.t.overnight, under nitrogen. After 23 h the reaction mixture was dilutedwith EtOAc and saturated aqueous NaHCO₃ and well mixed. The layers wereseparated and the aqueous layer extracted with EtOAc (1×). The combinedorganic layers were washed with H₂O (1×), brine (1×), dried (Na₂SO₄) andsolvent removed under vacuum. The crude product was purified by columnchromatography on silica gel using DCM:MeOH 100:0 to 93:7 and thematerial recolumned using DCM:MeOH 99:1 to 94:6 to give 11 (Compound No.81, 118 mg, 47%, HPLC purity: 98.0%) as a pale yellow foam. ¹H NMR δ(400 MHz, DMSO-d₆) 8.43-8.41 (m, 1H), 8.22 (s, 1H), 7.96 (d, J=8.4 Hz,1H), 7.83 (d, J=8.4 Hz, 1H), 7.73-7.66 (m, 1H), 7.61-7.56 (m, 1H),7.51-7.45 (m, 2H), 7.22-7.17 (m, 1H), 7.10 (s, 1H), 6.97 and 6.92 (2s,1H), 6.56 (t, J=6.0 Hz, 1H), 5.46 (dd, J=9.1, 6.2 Hz, 1H), 4.42-4.20 (m,4H), 4.05-3.76 (m, 7H), 3.80 and 3.79 (2s, 3H), 3.52-3.47 (m, 1H),3.38-3.11 (m, 4H), 2.09-1.99 (m, 1H), 1.94-1.88 (m, 3H), 1.77-1.74 (m,2H), 1.65-1.62 (m, 2H), 1.48-1.42 (m, 2H), 1.35-1.23 (m, 1H), 1.15-1.10(m, 3H), 9H partially obscured by DMSO. HRMS m/z 969.3070 [(M+Na)⁺ calcdfor C₄₇H₅₅ClN₆NaO₉S₂ 969.3053].

A mixture of 10 (162 mg, 0.267 mmol), 66d (178 mg, 0.418 mmol, freshlymade by the procedure mentioned above), EDCI.HCl (184 mg, 0.960 mmol)and TsOH (11 mg, 0.0639 mmol) in dry DMA (5 mL) was stirred at r.t.overnight, under nitrogen. After 18.5 h the reaction mixture was dilutedwith EtOAc and H₂O and well mixed. The layers were separated and theorganic layer washed with saturated aqueous NaHCO₃ (1×), H₂O (1×), brine(1×), dried (Na₂SO₄) and solvent removed under vacuum. The crude productwas purified by column chromatography on silica gel using EtOAc:MeOH100:0 to 95:5 to give a yellow residue. This was further purified bypreparative HPLC (column: Synergi-MAX RP 4μ, 21.20×250 mm; flow rate: 12mL/min; mobile phase: solvent A: H₂O/ammonium formate buffer pH 3.45,solvent B: MeCN/H₂O 90:10; method: gradient, solvent A:solvent B 90:10to 10:90 to 0:100 over 30 min) to give compound 12 (89.3 mg, 33%, HPLCpurity: 99.5%) as a white foam. ¹H NMR δ (400 MHz, DMSO-d₆) 8.56 (s,1H), 8.43-8.41 (m, 1H), 8.04 (d, J=8.2 Hz, 1H), 7.92 (d, J=8.4 Hz, 1H),7.73-7.67 (m, 1H), 7.60-7.56 (m, 1H), 7.51-7.45 (m, 2H), 7.22-7.17 (m,1H), 7.10 (s, 1H), 6.98 and 6.92 (2s, 1H), 6.55 (t, J=5.6 Hz, 1H),5.47-5.44 (m, 1H), 4.40-4.36 (m, 1H), 4.30-4.19 (m, 3H), 4.04-3.86 (m,4H), 3.86-3.75 (m, 1H), 3.80 and 3.79 (2s, 3H), 3.52-3.46 (m, 1H),3.38-3.22 (m, 3H), 3.21-3.15 (m, 1H), 2.09-1.99 (m, 1H), 1.94-1.85 (m,3H), 1.78-1.74 (m, 2H), 1.69-1.60 (m, 2H), 1.55-1.40 (m, 2H), 1.47 and1.47 (2s, 18H), 1.28-1.23 (m, 1H), 1.15-1.10 (m, 3H). HRMS m/z 1035.3162[(M+Na)⁺ calcd for C₄₉H₆₂ClN₄NaO₁₁PS₂ 1035.3175].

A mixture of 12 (84.0 mg, 0.0829 mmol) and TFA (1 mL) in dry DCM (2 mL)was stirred at r.t. for 40 mins. The solvent was then removed undervacuum at 25° C. to give a green residue. The residue was dissolved inDCM, the solution diluted with EtOAc and the DCM removed under vacuum togive a white solid and the remaining solvent decanted. This process wasrepeated and the resulting solid was triturated with EtOAc and dried togive compound 13 (Compound No. 82, 43.8 mg, 59%, HPLC purity: 93.8%) asa white solid. ¹H NMR δ (400 MHz, DMSO-d₆) 8.47 (s, 1H), 8.44-8.42 (m,1H), 8.08 (d, J=8.3 Hz, 1H), 7.90 (d, J=8.3 Hz, 1H), 7.74-7.68 (m, 1H),7.58-7.54 (m, 1H), 7.51-7.48 (m, 1H), 7.47-7.43 (m, 1H), 7.22-7.18 (m,1H), 7.10 (s, 1H), 6.98 and 6.93 (2s, 1H), 5.46 (d, J=9.5 Hz, 1H),4.39-4.18 (m, 4H), 4.04-3.95 (m, 3H), 3.90-3.85 (m, 1H), 3.84-3.76 (m,1H), 3.80 and 3.80 (2s, 3H), 3.52-3.47 (m, 1H), 3.40-3.27 (m, 3H),3.21-3.15 (m, 1H), 2.10-2.02 (m, 1H), 1.94-1.88 (m, 3H), 1.78-1.74 (m,2H), 1.69-1.60 (m, 2H), 1.48-1.42 (m, 2H), 1.35-1.23 (m, 1H), 1.16-1.10(m, 3H), 3H not observed. HRMS m/z 923.1938 [(M+Na)⁺ calcd forC₄₁H₄₆ClN₄NaO₁₁PS₂ 923.1923].

A mixture of 10 (45.0 mg, 0.0743 mmol), 67d (24.3 mg, 0.0899 mmol,freshly made by the procedure mentioned above), EDCI.HCl (42.7 mg, 0.223mmol) and TsOH (3 mg, 0.0174 mmol) in dry DMA (3 mL) was stirred at r.t.under nitrogen for 5 h. Additional portions of 67d (24.3 mg, 0.0899mmol) and EDCI.HCl (16.0 mg, 0.0835 mmol) were added to the mixture andthe reaction stirred at r.t. overnight. After 22 h the reaction mixturewas diluted with EtOAc and washed with H₂O (2×), brine (1×), dried(Na₂SO₄) and solvent removed under vacuum. The crude product waspurified by column chromatography on silica gel using EtOAc to give agreen powder. This was further purified carrying out columnchromatography on silica gel using EtOAc a second time to give compound14 (Compound No. 83, 8.3 mg, 13.5%, HPLC purity: 81.2%) as a beigesolid. ¹H NMR δ (400 MHz, DMSO-d₆) 10.33 (s, 1H), 8.43-8.42 (m, 1H),8.07 (d, J=8.1 Hz, 1H), 7.98 (s, 1H), 7.77 (d, J=8.4 Hz, 1H), 7.74-7.67(m, 1H), 7.50-7.46 (m, 2H), 7.33-7.29 (m, 1H), 7.24-7.18 (m, 1H), 7.10(s, 1H), 6.98 and 6.92 (2s, 1H), 6.56 (t, J=6.0 Hz, 1H), 5.47-5.44 (m,1H), 4.33-4.21 (m, 2H), 4.15-4.13 (m, 2H), 4.05-3.93 (m, 3H), 3.90-3.75(m, 2H), 3.80 and 3.79 (2s, 3H), 3.52-3.47 (m, 1H), 3.38-3.13 (m, 4H),2.10-1.99 (m, 1H), 1.94-1.89 (m, 3H), 1.77-1.74 (m, 2H), 1.66-1.62 (m,2H), 1.52-1.41 (m, 2H), 1.32-1.24 (m, 1H), 1.15-1.10 (m, 3H). HRMS m/z843.2258 [(M+Na)⁺ calcd for C₄₁H₄₅ClN₄NaO₈S₂ 843.2260].

Example 29(1S)-1-(chloromethyl)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(chloromethyl)-5-[(6-methyl-β-D-glucopranuronosyl)oxy]-1,2-dihydro-3H-benzo[e]indol-3-yl}-3-oxo-1-propenyl)-2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]phenyl}-2-propenoyl)-1,2-dihydro-3H-benzo[e]indol-5-ylmethyl β-D-glucopyranosiduronate (Compound No. 84, Table 4, FIG. 44)

Trichloroacetimidate (1) was prepared according to literatureprocedures: L. Lázár, E. Mezö, M. Herczeg, A. Lipták, S. Antus, A.Borbás, Tetrahedron 2012, 68, 7386-7399; L. Tietze, H. Schuster, K.Schmuck, I. Schuberth, F. Alves, Bioorg. & Med. Chem. 2008, 16,6312-6318.

A suspension of trichloroacetimidate (1) (360 mg, 0.75 mmol), phenol-CBI(2, compound 51a in first patent filing) (200 mg, 0.60 mmol) andactivated molecular sieves 4 Å (1 g) in anhydrous CH₂Cl₂ (20 mL) wasstirred at RT for 1 h. The mixture was cooled to −10° C., then BF₃.OEt₂(40 μl, 0.3 mmol) was added dropwise. The temperature was kept between−10° C. and −5° C. for 1 h, it was then stirred at 0° C. for 30 min.BF₃—OEt₂ (0.24 mL, 1.8 mmol) was subsequently added dropwise at 0° C.,the temperature allowed to raise to RT and it was stirred for 2 h. Thesuspension was then filtered over celite and the solvent evaporated togive crude CBI-Glucuronide (3) which was used in the next step withoutfurther purification. A solution of amine (3) and bis-acid (4, compound66h in first patent filing) (126 mg, 0.24 mmol) in anhydrous DMA (4 mL)was cooled to 0° C. pTsOH (17 mg, 0.096 mmol) and EDCI-HCl (276 mg, 1.44mmol) were then added, the temperature was allowed to raise to RT and itwas stirred for 16 h. The solvent was removed under reduced pressure andthe residue purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 0-2%)then (SiO₂, CH₂Cl₂/MeOH 1-2%) to give(1S)-1-(chloromethyl)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(chloromethyl)-5-[(2,3,4-tri-O-acetyl-6-methyl-β-D-glucopyranuronosyl)oxy]-1,2-dihydro-3H-benzo[e]indol-3-yl}-3-oxo-1-propenyl)-2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]phenyl}-2-propenoyl)-1,2-dihydro-3H-benzo[e]indol-5-ylmethyl 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronate (5) (127 mg, 27%) asa yellow solid. ¹H NMR (300 MHz, [(CD₃)₂SO]) δ 10.04 (s, 1H, NH), 8.38(br s, 2H), 8.11 (d, J=8.1 Hz, 1H), 7.97 (d, J=8.5 Hz, 2H), 7.93 (d,J=8.2 Hz, 2H), 7.83-7.88 (m, 4H), 7.78 (d, J=7.6 Hz, 1H), 7.68-7.72 (m,3H), 7.59 (t, J=7.5 Hz, 2H), 7.45-7.48 (m, 3H), 7.38 (t, J=7.4 Hz, 2H),7.25-7.29 (m, 4H), 5.85 (d, J=7.7 Hz, 1H), 5.83 (d, J=7.8 Hz, 1H),5.62-5.65 (m, 2H), 5.32 (t, J=8.7 Hz, 2H), 5.14 (t, J=9.6 Hz, 2H), 4.78(t, J=8.2 Hz, 2H), 4.55 (m, 4H), 4.31-4.38 (m, 4H), 4.22 (t, J=6.9 Hz,1H), 4.01-4.03 (m, 2H), 3.91-3.96 (m, 2H), 3.65 (s, 3H), 3.63 (s, 3H),3.36-3.38 (m, 2H), 2.59-2.64 (m, 2H), 1.98-2.03 (m, 18H); LC-MS (ESI)Calcd for C₈₂H₇₉Cl₂N₄O₂₅ (M+H)⁺ m/z 1591.4. found m/z 1591.4; Calcd forC₈₂H₇₈Cl₂N₄O₂₅Na (M+Na)⁺ m/z 1613.4. found m/z 1613.4.

A solution of derivative (5) (110 mg, 0.07 mmol) and piperidine (468 μL,0.7 mmol) in anhydrous DMF (5 mL) was stirred at RT for 30 min. Thesolvent was removed under reduced pressure at RT and the residue wastriturated with cold Et₂O, to give crude(1S)-3-{(2E)-3-[2-[(3-aminopropanoyl)amino]-4-((1E)-3-{(1S)-1-(chloromethyl)-5-[(2,3,4-tri-O-acetyl-6-methyl-3-D-glucopyranuronosyl)oxy]-1,2-dihydro-3H-benzo[e]indol-3-yl}-3-oxo-1-propenyl)phenyl]-2-propenoyl}-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indol-5-ylmethyl 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronate (6) (86 mg, 90%)which was used in the next step without further purification. ¹H NMR(300 MHz, [(CD₃)₂SO] δ 8.38 (br s, 2H), 8.10 (d, J=8.4 Hz, 1H), 7.98 (d,J=8.4 Hz, 2H), 7.94 (d, J=8.4 Hz, 2H), 7.90 (s, 1H), 7.88 (d, J=15.1 Hz,1H), 7.76 (d, J=7.6 Hz, 1H), 7.70 (d, J=15.1 Hz, 1H), 7.59 (t, J=7.9 Hz,2H), 7.47 (t, J=7.9 Hz, 2H), 7.27 (dd, J=3.4, 15.3 Hz, 2H), 5.85 (d,J=7.8 Hz, 1H), 5.84 (d, J=7.8 Hz, 1H), 5.63-5.68 (m, 2H), 5.32 (dd,J=7.8, 9.6 Hz, 2H), 5.14 (dt, J=1.5, 9.6 Hz, 2H), 4.79 (d, J=9.6 Hz,2H), 4.56-4.60 (m, 4H), 4.29-4.41 (m, 2H), 4.02-4.04 (m, 2H), 3.92-3.97(m, 2H), 3.67 (s, 6H), 2.93 (t, J=6.4 Hz, 2H), 2.03-2.04 (m, 18H), 2Hunder DMSO peak, NH and NH₂ not observed.

To a stirred solution of amine (6) (110 mg, 0.08 mmol) in a (1:1)mixture of MeOH/CH₂Cl₂ (10 mL) was added dropwise a solution of NaOMe(8.7 mg, 0.16 mmol) in MeOH (1 mL) at 0° C., and the reaction mixturewas stirred at 0° C. for 2 h. AcOH (8 drops) was then added and thesolvent was removed under reduced pressure at RT and dried under highvacuum to give(1S)-3-{(2E)-3-[2-[(3-aminopropanoyl)amino]-4-((1E)-3-{(1S)-1-(chloromethyl)-5-[(6-methyl-3-D-glucopyranuronosyl)oxy]-1,2-dihydro-3H-benzo[e]indol-3-yl}-3-oxo-1-propenyl)phenyl]-2-propenoyl}-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indol-5-ylmethyl β-D-glucopyranosiduronate (7) a an orange solid which was used inthe next step without further purification. To a stirred solution ofamine (7) and N-succinimidyl 6-maleimidohexanoate (24 mg, 0.077 mmol) inanhydrous DMF (5 mL) was added DIEA (78 μL, 0.1 mmol) and the mixturewas stirred at RT overnight. The solvent was then removed under reducedpressure at RT and the residue was purified by column chromatography(SiO₂, EtOAc/MeOH 10-20%) then twice (SiO₂, EtOAc/MeOH 15%) to give(1S)-1-(chloromethyl)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(chloromethyl)-5-[(6-methyl-3-D-glucopyranuronosyl)oxy]-1,2-dihydro-3H-benzo[e]indol-3-yl}-3-oxo-1-propenyl)-2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]phenyl}-2-propenoyl)-1,2-dihydro-3H-benzo[e]indol-5-ylmethyl 3-D-glucopyranosiduronate 8 (Compound No. 84) (31 mg, 34%) as ayellow solid. HPLC purity 95.9%; ¹H NMR (300 MHz, [(CD₃)₂SO] δ 10.01 (s,1H), 8.32 (s, 2H), 8.31 (d, J=8.6 Hz, 2H), 8.09 (d, J=8.2 Hz, 1H),7.89-7.95 (m, 3H), 7.84 (t, J=7.6 Hz, 2H), 7.77 (d, J=8.0 Hz, 1H), 7.70(d, J=15.1 Hz, 1H), 7.58 (t, J=7.9 Hz, 2H), 7.43 (t, J=8.0 Hz, 2H), 7.27(d, J=15.6 Hz, 2H), 6.96 (s, 2H), 5.67 (d, J=5.3 Hz, 1H), 5.66 (d, J=5.3Hz, 1H), 5.45 (d, J=5.6 Hz, 2H), 5.35 (d, J=4.6 Hz, 2H), 5.16 (d, J=7.5Hz, 2H), 4.52-4.62 (m, 4H), 4.33 (br s, 2H), 3.99-4.04 (m, 4H), 3.93(dd, J=7.4, 10.5 Hz, 2H), 3.68 (s, 3H), 3.67 (s, 3H), 3.37-3.48 (m, 8H),2.57-2.61 (m, 2H), 2.09 (t, J=7.3 Hz, 2H), 1.41-1.54 (m, 4H), 1.14-1.23(m, 4H); HRMS (ESI) Calcd for C₆₅H₆₇Cl₂N₅NaO₂₀ (M+Na⁺) m/z 1330.3649.found m/z 1330.3600.

Example 30(S)-(1-methyl-1H-pyrrole-2,5-diyl)bis(((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)methanone)(Compound No. 15, table 1, FIG. 45)

A mixture of 1 (500 mg, 3.22 mmol) and N,N-Dimethylformamidedi-tert-butyl acetal (2, 5.24 g, 25.77 mmol) in NMP (10 mL) was heatedup to 100° C. and stirred overnight. Most volatile components wereremoved under reduced pressure and the resultant residue wasredistributed between ethyl acetate and water. The aqueous phase wasextracted with ethyl acetate three times. The combined organic extractswere washed with water followed by brine, dried over anhydrous Na₂SO₄,and filtered through a pad of silica gel. The solvent was removed and 3was obtained as a pale crystalline solid (331 mg, 38%); ¹H NMR (DMSO) δ12.26 (s, 1H), 6.68 (d, J=2.0 Hz, 2H), 1.51 (s, 18H) ppm. HRMS (ESI)found m/z 290.1362 (M+Na). C₁₄H₂₁NNaO₄ requires 290.1363.

A mixture of 3 (50 mg, 0.18 mmol), K₂CO₃ (52 mg, 0.37 mmol), MeI (0.12mL, 1.87 mmol) and tetrabutylamonium iodide (3.5 mg, 0.0094 mmol) inMeCN (1 mL) and water (0.01 mL) was heated at 35-40° C. and stirred for3 days. The reaction mixture was redistributed between ethyl acetate andwater. The aqueous phase was extracted with ethyl acetate three times.The combined organic extracts were washed with water followed by brine,dried over anhydrous Na₂SO₄, and filtered through a pad of silica gel.The solvent was removed and 4 was obtained as a pale oil, which became acolourless crystalline solid in hours (45 mg, 86%); ¹H NMR (CDCl₃) δ6.77 (s, 2H), 4.21 (s, 3H), 1.56 (s, 18H) ppm. HRMS (ESI) found m/z304.1512 (M+Na). C₁₅H₂₃NNaO₄ requires 304.1519.

To a solution of 4 (45 mg, 0.16 mmol) in DCM (1 mL) at room temperaturewas added TFA (0.5 mL, 6.49 mmol). The mixture was stirred for 3 h togive a pink solution. All volatile components were pumped off and theresultant residue was triturated with petroleum ether to give 5 as apink solid (25 mg, 93%). ¹H NMR (DMSO) δ 12.81 (s, 2H), 6.80 (s, 2H),4.15 (s, 3H) ppm. HRMS (ESI negative) found m/z 168.0306 (M−H). C₇H₆NO₄requires 168.0302.

To a solution of Boc-CBI-OH (Comp 51a, 130 mg, 0.39 mmol) in DCM (2 mL)at room temperature was added 4N HCl in dioxane (2 mL). The mixture wasstirred for 2.5 h. All volatile components were pumped off and theresultant residue (6) was used directly as it was.

A mixture of 6 (made above), 5 (22 mg, 0.13 mmol), EDCI hydrochloride(150 mg, 0.78 mmol) and toluenesulfonic acid (2.2 mg, 0.013 mmol) in DMA(2 mL) was stirred at room temperature overnight. Most of the solventwas pumped off and the resultant residue was redistributed between ethylacetate and water. The aqueous phase was extracted with ethyl acetatethree times. The combined organic extracts were washed with waterfollowed by brine, dried over anhydrous Na₂SO₄, and filtered through apad of Celite. The solvent was removed and the resultant residue waspurified by silica gel column chromatography using a mixture of MeOH andDCM (v/v 2%) as eluent to give 7 (Compound No. 15, Table 1) as anoff-white solid (60 mg, 77%). ¹H NMR (DMSO) δ 10.43 (s, 2H), 8.12 (d,J=8.3 Hz, 2H), 7.83 (d, J=8.3 Hz, 2H), 7.73 (br s, 2H), 7.52 (dd, J=1.0,8.0 Hz, 2H), 7.37 (dd, J=0.4, 8.0 Hz, 2H), 6.77 (s, 2H), 4.62-4.57 (m,2H), 4.31-4.27 (m, 2H), 4.10-4.07 (m, 2H), 4.02-4.00 (m, 2H), 3.88-3.85(m, 5H) ppm. HRMS (ESI) found m/z 622.1255 (M+Na). C₃₃H₂₇Cl₂N₃NaO₄requires 622.1271.

Example 31N-(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)phenyl)acetamide(Compound No. 16, Table 1, FIG. 46)

To a solution of 1 (66f, 500 mg, 1.45 mmol) in THF (5 mL) and pyridine(5 mL) in an ice bath was added acetyl chloride (0.50 mL, 7.03 mmol).The mixture was allowed to warm up to room temperature and stirredovernight. All volatile components were pumped off and the resultantresidue was redistributed between ethyl acetate and aqueous sodiumbicarbonate. The aqueous phase was extracted with ethyl acetate threetimes. The combined organic extracts were washed with water followed bybrine, dried over anhydrous Na₂SO₄, and filtered through a pad ofCelite. The solvent was removed and the resultant residue was purifiedby silica gel column chromatography using a mixture of ethyl acetate andpetroleum ether (v/v 1:2) as eluent to give 2 as an off-white solid (475mg, 85%); ¹H NMR (CDCl₃) δ 8.00 (s, 1H), 7.70 (d, J=15.8 Hz, 1H),7.55-7.51 (m, 2H), 7.31 (apparent d, J=8.6 Hz, 2H), 6.40 (d, J=16.0 Hz,1H), 6.37 (d, J=15.8 Hz, 1H), 2.25 (s, 3H), 1.54 (s, 9H), 1.53 (s, 9H)ppm. HRMS (ESI) found m/z 410.1921 (M+Na). C₂₂H₂₉NNaO₅ requires410.1938.

To a solution of 2 (470 mg, 1.21 mmol) in DCM (5 mL) at room temperaturewas added TFA (2.5 mL, 32.40 mmol). The mixture was stirred for 3 h togive a white suspension. More DCM was added to precipitate out moresolid, which was collected by filtration and washed with ethyl acetateand petroleum ether. 3 was obtained as a white solid (290 mg, 87%). ¹HNMR (DMSO) δ 12.40 (br s, 2H), 9.89 (s, 1H), 7.85 (d, J=8.3 Hz, 1H),7.70 (apparent d, J=16.0 Hz, 2H), 7.57-7.53 (m, 2H), 6.54 (d, J=15.9 Hz,1H), 6.53 (d, J=16.0 Hz, 1H), 2.09 (s, 3H) ppm. HRMS (ESI) found m/z298.0672 (M+Na). C₁₄H₁₃NNaO₅ requires 298.0686.

To a solution of Boc-CBI-OH (Comp 51a, 291 mg, 0.87 mmol) in DCM (3 mL)at room temperature was added 4N HCl in dioxane (3 mL). The mixture wasstirred for 2.5 h. All volatile components were pumped off and theresultant residue (4) was used directly as it was.

A mixture of 4 (made above), 3 (80 mg, 0.29 mmol), EDCI hydrochloride(334 mg, 1.74 mmol) and toluenesulfonic acid (5 mg, 0.029 mmol) in DMA(3 mL) was stirred at room temperature overnight. All the volatilecomponents were pumped off and the resultant residue was triturated withmethanol several times to give crude product (123 mg), which wasdissolved in THF and precipitated by the addition of MeOH to give 5(Compound No. 16, Table 1) as a yellow solid (96 mg, 47%, HPLC purity97%); ¹H NMR (DMSO) δ 10.43 (s, 2H), 9.97 (s, 1H), 8.12-8.07 (m, 5H),7.85-7.81 (m, 4H), 7.74 (apparent d, J=6.7 Hz, 1H), 7.68 (d, J=15.4 Hz,1H), 7.73 (t, J=7.4 Hz, 2H), 7.35 (t, J=7.5 Hz, 2H), 7.26 (dd, J=3.2,15.3 Hz, 2H), 4.58-4.46 (m, 4H), 4.28-4.22 (m, 2H), 4.05-3.98 (m, 2H),3.88-3.82 (m, 2H), 2.15 (s, 3H) ppm. HRMS (ESI) found m/z 728.1662(M+Na). C₄₀H₃₃Cl₂N₃NaO₅ requires 728.1689.

Example 32(S,2E,2′E)-3,3′-(2-methoxy-1,4-phenylene)bis(1-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)prop-2-en-1-one)(Compound No. 17, Table 1, FIG. 47)

To a solution of 1 (2.50 g, 13.30 mmol) in dry THF (12 mL) at −30 to−40° C. in a dry ice-MeCN bath was added boron trifluoride diethyletherate (BF₃.Et₂O, 4.92 mL, 39.90 mmol) dropwise under N₂. After themixture was stirred at −30° C. for 10 min, ^(t)BuONO (2.39 mL, 19.94mmol) was added dropwise. The reaction mixture was allowed to warm up toroom temperature and stirred for 1.5 h to give a suspension. Petroleumether (50 mL) was added to give more precipitate. The supernatant wasremoved by decantation and the solid left was washed with petroleumether to afford a white solid. This solid was dissolved in dry MeCN (20mL) and cooled in an ice bath. KI (11.00 g, 66.26 mmol) and I₂ (6.00 g,23.64 mmol) were added. The reaction mixture was stirred at roomtemperature for 4 h before saturated Na₂S₂O₃ solution (50 mL) was addedto quench the reaction. The mixture was extracted with ethyl acetatethree times. The combined organic extracts were washed with waterfollowed by brine, dried over anhydrous Na₂SO₄, and filtered through apad of Celite. The solvent was removed and the resultant residue waspurified by silica gel column chromatography using a mixture of ethylacetate and petroleum ether (v/v 1:9) as eluent to give 2 as a palesolid (2.83 g, 71%); ¹H NMR (CDCl₃) δ 7.50 (d, J=8.5 Hz, 1H), 7.16 (d,J=2.2 Hz, 1H), 6.84 (dd, J=2.2, 8.5 Hz, 1H), 5.39 (s, 1H) ppm.

A mixture of 2 (500 mg, 1.67 mmol), tert-butyl acrylate (0.728 mL, 5.02mmol), palladium (II) acetate (7.5 mg, 0.033 mmol) and tri-ortho-tolylphosphine (41 mg, 0.13 mmol) in redistilled triethylamine (5 mL) washeated at reflux overnight under N₂ to give a dark grey suspension. Allvolatile components were pumped off. The resultant residue was dissolvedin ethyl acetate and the precipitate was filtered off. The filtrate wasevaporated and the residue obtained was purified by columnchromatography using a mixture of ethyl acetate and petroleum ether (v/v1:6) as eluent to give 3 (160 mg, 28%) as an off-white solid. ¹H NMR(DMSO) δ 10.43 (s, 1H), 7.75 (d, J=16.4 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H),7.45 (d, J=16.0 Hz, 1H), 7.18 (d, J=8.0 Hz, 1H), 7.07 (d, J=0.8 Hz, 1H),6.56 (d, J=16.4 Hz, 1H), 6.41 (d, J=15.6 Hz, 1H), 1.483 (s, 9H), 1.478(s, 9H) ppm. HRMS (ESI) found m/z 369.1687 (M+Na). C₂₀H₂₆NaO₅ requires369.1672.

To a solution of 3 (160 mg, 0.46 mmol) in DMF (2 mL) was added K₂CO₃(254 mg, 1.84 mmol) and MeI (0.28 mL, 4.50 mmol). The mixture wasstirred at room temperature overnight and the precipitate was filteredoff. The resultant filtrate was washed with water followed by brine,dried over anhydrous Na₂SO₄, and filtered through a pad of Celite. Thesolvent was removed and the resultant residue was purified by silica gelcolumn chromatography using a mixture of ethyl acetate and petroleumether (v/v 1:10) as eluent to give 4 as a colourless oil (72 mg, 43%);¹H NMR (CDCl₃) δ 7.87 (d, J=16.2 Hz, 1H), 7.54 (d, J=15.9 Hz, 1H), 7.49(d, J=8.0 Hz, 1H), 7.10 (dd, J=1.3, 8.0 Hz, 1H), 7.00 (d, J=1.2 Hz, 1H),6.47 (d, J=16.1 Hz, 1H), 6.38 (d, J=15.9 Hz, 1H), 3.91 (s, 3H), 1.539(s, 9H), 1.533 (s, 9H) ppm. HRMS (ESI) found m/z 383.1838 (M+Na).C₂₁H₂₈NaO₅ requires 383.1829.

To a solution of 4 (70 mg, 0.19 mmol) in DCM (2 mL) at room temperaturewas added TFA (1 mL, 12.98 mmol). The mixture was stirred for 2.5 h togive a white suspension. All volatile components were pumped off and theresultant residue was triturated with DCM and ethyl acetate to give 5 asa white solid (41 mg, 85%). ¹H NMR (DMSO) δ 12.41 (br s, 2H), 7.80 (d,J=16.2 Hz, 1H), 7.72 (d, J=8.0 Hz, 1H), 7.58 (d, J=16.0 Hz, 1H), 7.41(d, J=1.2 Hz, 1H), 7.30 (dd, J=1.0, 8.1 Hz, 1H), 6.47 (d, J=16.0 Hz,1H), 6.38 (d, J=16.1 Hz, 1H), 3.92 (s, 3H) ppm. HRMS (ESI) found m/z271.0573 (M+Na). C₁₃H₁₂NaO₅ requires 271.0577.

To a solution of Boc-CBI-OH (Comp 51a, 161 mg, 0.48 mmol) in DCM (2 mL)at room temperature was added 4N HCl in dioxane (2 mL). The mixture wasstirred for 2.5 h. All volatile components were pumped off and theresultant residue (6) was used directly as it was.

A mixture of 6 (made above), 5 (40 mg, 0.16 mmol), EDCI hydrochloride(185 mg, 0.97 mmol) and toluenesulfonic acid (2.8 mg, 0.016 mmol) in DMA(1 mL) was stirred at room temperature overnight. All the volatilecomponents were pumped off and the resultant residue was triturated withmethanol to give a yellow solid, which was dissolved in THF andprecipitated by the addition of methanol to afford 7 (Compound No. 17,Table 1) as a yellow solid (45 mg, 41%, HPLC purity 98%); ¹H NMR (DMSO)δ 10.43 (s, 2H), 8.12-8.10 (m, 4H), 8.00-7.95 (m, 2H), 7.85-7.80 (m,2H), 7.72 (d, J=15.4 Hz, 1H), 7.54-7.50 (m, 4H), 7.37-7.26 (m, 4H),4.57-4.45 (m, 4H), 4.28-4.22 (m, 2H), 4.00-3.99 (m, 5H), 3.90-3.83 (m,2H) ppm. HRMS (ESI) found m/z 701.1596 (M+Na). C₃₉H₃₂Cl₂N₂NaO₅ requires701.1580.

Example 33(S,2E,2′E)-3,3′-(1-methyl-1H-pyrrole-2,5-diyl)bis(1-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)prop-2-en-1-one)(Compound No. 18 Table 1, FIG. 48)

1 was prepared using a literature method (Ref. Russ J Org Chem, 2007,43, 855-860.)

2

A mixture of 1 (50 mg, 0.41 mmol), K₂CO₃ (112 mg, 0.81 mmol), MeI (0.25mL, 4.06 mmol) and tetrabutylamonium iodide (7.5 mg, 0.020 mmol) in MeCN(2 mL) and water (0.02 mL) was heated at 40° C. and stirred for 3 h.Most volatile components were removed under reduced pressure and theresultant residue was redistributed between ethyl acetate and water. Theaqueous phase was extracted with ethyl acetate three times. The combinedorganic extracts were washed with water followed by brine, dried overanhydrous Na₂SO₄, and filtered through a pad of silica gel. The solventwas removed and 2 was obtained as a white solid (47 mg, 84%); with ¹HNMR spectrum identical to that reported (Ref. C. E. Loader, G. H.Barnett and H. J. Anderson, Can. J. Chem., 1982, 60, 383.)

4

A mixture of 2 (40 mg, 0.29 mmol) and methyl(triphenylphosphoranylidene)acetate (3, 400 mg, 1.20 mmol) in DCM (3 mL)was stirred for two days to give a yellow solution. The mixture waspurified by silica gel column chromatography using gradient mixtures ofethyl acetate and petroleum ether (v/v=1:5, 1:4 and 1:3) as eluent togive 4 as a yellow solid (63 mg, 87%); ¹H NMR (CDCl₃) δ 7.61 (d, J=15.6Hz, 2H), 6.70 (s, 2H), 6.26 (d, J=15.6 Hz, 2H), 3.79 (s, 6H), 3.73 (s,3H) ppm. HRMS (ESI) found m/z 272.0898 (M+Na). C₁₃H₁₅NNaO₄ requires272.0893.

5

A mixture of 4 (60 mg, 0.24 mmol) and KOH (100 mg, 1.78 mmol) in EtOH (2mL) and THF (1 mL) was stirred at 70° C. for 2h. The mixture wasevaporated under reduced pressure to dryness. The resultant residue wasdissolved in water and acidified with 1N HCl till pH 5 to give a yellowprecipitate, which was collected by filtration, and then washed withwater and petroleum ether to give 5 as a yellow solid (51 mg, 96%); ¹HNMR (DMSO) δ 12.20 (s, 2H), 7.54 (d, J=15.6 Hz, 2H), 6.89 (s, 2H), 6.30(d, J=15.6 Hz, 2H), 3.70 (s, 3H) ppm. HRMS (ESI) found m/z 244.0590(M+Na). C₁₁H₁₁NNaO₄ requires 244.0580.

7

To a solution of Boc-CBI-OH (Comp 51a, 200 mg, 0.60 mmol) in DCM (2 mL)at room temperature was added 4N HCl in dioxane (10 mL). The mixture wasstirred for 2 h. All volatile components were pumped off and theresultant residue (6, comp 67d) was used directly as it was.

A mixture of 6 (made above), 5 (49 mg, 0.22 mmol), EDCI hydrochloride(255 mg, 1.33 mmol) and toluenesulfonic acid (3.8 mg, 0.022 mmol) in DMA(2 mL) was stirred at room temperature overnight. The solvent wasremoved and the resultant residue was purified by silica gel columnchromatography using a mixture of MeOH and ethyl acetate (v/v 3%) andthe crude product was triturated with ethyl acetate to give 7 as anorange solid (60 mg, 41%, HPLC 100%); ¹H NMR (DMSO) δ 10.41 (s, 2H),8.11 (d, J=8.2 Hz, 4H), 7.81 (d, J=8.3 Hz, 2H), 7.70 (d, J=14.9 Hz, 2H),7.50 (t, J=7.4 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 7.12 (s, 2H), 7.01 (d,J=14.9 Hz, 2H), 4.49-4.42 (m, 4H), 4.24-4.19 (m, 2H), 4.01-3.97 (m, 2H),3.85-3.82 (m, 5H) ppm. HRMS (ESI negative) found m/z 650.1600 (M−H).C₃₇H₃₀Cl₂N₃O₄ requires 650.1619.

Example 34(S)-3,3′-(2-methoxy-1,4-phenylene)bis(1-((S)-1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)prop-2-yn-1-one)(Compound 19, table 1, FIG. 49)

2

Compound 2 was synthesised by a modified procedure based on thatdescribed in US2007/49758. To a suspension of 1 (5.50 g, 25.00 mmol) andsilver (I) acetate (6.26 g, 37.5 mmol) in DCM (100 mL) was addeddropwise a solution of iodine (6.98 g, 27.50 mmol) in DCM (150 mL). Theresulting mixture was stirred overnight at room temperature, and thenfiltered through a pad of Celite. The filtrate was evaporated and theresultant residue was purified by silica gel column chromatography usinga mixture of ethyl acetate and petroleum ether (v/v 1:10) as eluent togive 2 as a white solid (966 mg, 11%); mp 97-99° C. ¹H NMR (CDCl₃) δ7.342 (d, J=8.3 Hz, 1H), 7.339 (d, J=2.0 Hz, 1H), 7.00 (dd, J=2.0, 8.3Hz, 1H), 5.32 (s, 1H) ppm. ¹³C NMR (CDCl₃, 100.6 MHz) δ 155.70, 139.46(CH), 131.80 (CH), 124.44 (CH), 94.65, 85.52 ppm.

3

To a solution of 2 (270 mg, 0.78 mmol) in DMF (4 mL) was added K₂CO₃(162 mg, 1.17 mmol) and MeI (0.24 mL, 3.90 mmol). The mixture wasstirred at room temperature for 2 h. The mixture was filtered through apad of silica gel and the filtrate was evaporated to give 3 as acolourless oil (277 mg, 99%); ¹H NMR (CDCl₃) δ 7.45 (d, J=8.1 Hz, 1H),7.09 (d, J=1.8 Hz, 1H), 7.04 (dd, J=1.8, 8.1 Hz, 1H), 3.87 (s, 3H) ppm.

4

A mixture of 3 (274 mg, 0.76 mmol), tert-butyl propiolate (0.314 mL,2.28 mmol), copper (I) iodide (5.8 mg, 0.030 mmol), palladium (II)acetate (3.4 mg, 0.015 mmol) and triphenyl phosphine (12 mg, 0.046 mmol)in redistilled triethylamine (5 mL) was heated at 60° C. overnight underN₂ to give a dark-coloured suspension. All volatile components werepumped off. The resultant residue was stirred with DCM and theprecipitate was filtered off. The filtrate was evaporated and theresidue obtained was purified by silica gel column chromatography usinga mixture of DCM and petroleum ether (v/v=1:1) as eluent to give 4 (195mg, 72%) as an off-white solid. ¹H NMR (CDCl₃) δ 7.48 (d, J=7.9 Hz, 1H),7.13 (d, J=7.9 Hz, 1H), 7.06 (s, 1H), 3.89 (s, 3H), 1.545 (s, 9H) and1.514 (s, 9H) ppm. HRMS (ESI) found m/z 379.1510 (M+Na). C₂₁H₂₄NaO₅requires 379.1516.

5

To a solution of 4 (100 mg, 0.28 mmol) in DCM (2 mL) at room temperaturewas added TFA (1 mL, 12.98 mmol). The mixture was stirred for 2.5 h togive a white suspension. All volatile components were pumped off and theresultant residue was triturated with a mixture of DCM and petroleumether (v/v=1:1) to give 5 as a white solid (66 mg, 96%). ¹H NMR (DMSO) δ13.92 (br s, 2H), 7.61 (d, J=7.9 Hz, 1H), 7.37 (apparent s, 1H), 7.25(dd, J=1.1, 7.9 Hz, 1H), 3.90 (s, 3H) ppm.

7

To a solution of Boc-CBI-OH (Comp 51a, 267 mg, 0.80 mmol) in DCM (3 mL)at room temperature was added 4N HCl in dioxane (3 mL). The mixture wasstirred for 2.5 h. All volatile components were pumped off and theresultant residue (6) was used directly as it was.

A mixture of 6 (made above), 5 (65 mg, 0.27 mmol), EDCI hydrochloride(306 mg, 1.60 mmol) and toluenesulfonic acid (4.6 mg, 0.027 mmol) in DMA(1 mL) was stirred at room temperature overnight. All the volatilecomponents were pumped off and the resultant residue was triturated withmethanol to give a yellow solid, which was dissolved in THF andprecipitated by the addition of methanol to afford 7 as a yellow solid(140 mg, 78%, HPLC purity 99%); ¹H NMR (DMSO) δ 10.52 (s, 2H), 8.12 (d,J=8.4 Hz, 2H), 7.90-7.85 (m, 4H), 7.76 (d, J=7.9 Hz, 1H), 7.56-7.50 (m,3H), 7.41-7.37 (m, 3H), 4.63-4.49 (m, 4H), 4.29-4.21 (m, 2H), 4.09-4.06(m, 2H), 4.01 (s, 3H), 3.99-3.94 (m, 1H), 3.91-3.86 (m, 1H) ppm. HRMS(ESI) found m/z 697.1267 (M+Na). C₃₉H₂₈Cl₂N₂NaO₅ requires 697.1267.

Example 19 Preparation of Cysteine Engineered Antibodies for Conjugationby Reduction and Reoxidation

Under certain conditions, the cysteine engineered antibodies may be madereactive for conjugation with linker-drug intermediates of theinvention, such as those in Table 4, by treatment with a reducing agentsuch as DTT (Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.). Full length,cysteine engineered monoclonal antibodies (ThioMabs) expressed in CHOcells (Gomez et al (2010) Biotechnology and Bioeng. 105(4):748-760;Gomez et al (2010) Biotechnol. Prog. 26:1438-1445) were reduced, forexample with about a 50 fold excess of DTT overnight at room temperatureto reduce disulfide bonds which may form between the newly introducedcysteine residues and the cysteine present in the culture media.

Light chain amino acids are numbered according to Kabat (Kabat et al.,Sequences of proteins of immunological interest, (1991) 5th Ed., US Deptof Health and Human Service, National Institutes of Health, Bethesda,Md.). Heavy chain amino acids are numbered according to the EU numberingsystem (Edelman et al (1969) Proc. Natl. Acad. of Sci. 63(1):78-85),except where noted as the Kabat system. Single letter amino acidabbreviations are used.

Full length, cysteine engineered monoclonal antibodies (ThioMabs)expressed in CHO cells bear cysteine adducts (cystines) orglutathionylated on the engineered cysteines due to cell cultureconditions. To liberate the reactive thiol groups of the engineeredcysteines, the ThioMabs are dissolved in 500 mM sodium borate and 500 mMsodium chloride at about pH 8.0 and reduced with about a 50-100 foldexcess of 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride (Getzet al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly,Mass.) for about 1-2 hrs at 37° C. Alternatively, DTT can be used asreducing agent. The formation of inter-chain disulfide bonds wasmonitored either by non-reducing SDS-PAGE or by denaturing reverse phaseHPLC PLRP column chromatography. The reduced ThioMab is diluted andloaded onto a HiTrap SP FF column in 10 mM sodium acetate, pH 5, andeluted with PBS containing 0.3M sodium chloride, or 50 mM Tris-C1, pH7.5 containing 150 mM sodium chloride.

Disulfide bonds were reestablished between cysteine residues present inthe parent Mab by carrying out reoxidation. The eluted reduced ThioMabis treated with 15× or 2 mM dehydroascorbic acid (dhAA) at pH 7 for 3hours or for 3 hrs in 50 mM Tris-C1, pH 7.5, or with 2 mM aqueous coppersulfate (CuSO₄) at room temperature overnight. Other oxidants, i.e.oxidizing agents, and oxidizing conditions, which are known in the artmay be used. Ambient air oxidation may also be effective. This mild,partial reoxidation step forms intrachain disulfides efficiently withhigh fidelity. The buffer is exchanged by elution over Sephadex G25resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is checkedby determining the reduced antibody concentration from the absorbance at280 nm of the solution and the thiol concentration by reaction with DTNB(Aldrich, Milwaukee, Wis.) and determination of the absorbance at 412nm.

Liquid chromatography/Mass Spectrometric Analysis was performed on a TSQQuantum Triple Quadrupole™ mass spectrometer with extended mass range(Thermo Electron, San Jose Calif.). Samples were chromatographed on aPRLP-S®, 1000 A, microbore column (50 mm×2.1 mm, Polymer Laboratories,Shropshire, UK) heated to 75° C. A linear gradient from 30-40% B(solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile)was used and the eluent was directly ionized using the electrospraysource. Data were collected by the Xcalibur® data system anddeconvolution was performed using ProMass® (Novatia, LLC, New Jersey).Prior to LC/MS analysis, antibodies or drug conjugates (50 micrograms)were treated with PNGase F (2 units/ml; PROzyme, San Leandro, Calif.)for 2 hours at 37° C. to remove N-linked carbohydrates.

Hydrophobic Interaction Chromatography (HIC) samples were injected ontoa Butyl HIC NPR column (2.5 micron particle size, 4.6 mm×3.5 cm) (TosohBioscience) and eluted with a linear gradient from 0 to 70% B at 0.8ml/min (A: 1.5 M ammonium sulfate in 50 mM potassium phosphate, pH 7, B:50 mM potassium phosphate pH 7, 20% isopropanol). An Agilent 1100 seriesHPLC system equipped with a multi wavelength detector and Chemstationsoftware was used to resolve and quantitate antibody species withdifferent ratios of drugs per antibody.

Example 20 Conjugation of Linker-Drug Intermediates to Antibodies

After the reduction and reoxidation procedures of Example 19, theantibody is dissolved in PBS (phosphate buffered saline) buffer andchilled on ice. An excess, from about 1.5 molar to 20 equivalents of alinker-drug intermediate, including but not limited to 51-68 in Table 4,with a thiol-reactive functional group such as maleimido orbromo-acetamide, is dissolved in DMSO, diluted in acetonitrile andwater, and added to the chilled reduced, reoxidized antibody in PBS.After about one hour, an excess of maleimide is added to quench thereaction and cap any unreacted antibody thiol groups. The conjugationmixture may be loaded and eluted through a HiTrap SP FF column to removeexcess drug-linker intermediate and other impurities. The reactionmixture is concentrated by centrifugal ultrafiltration and the cysteineengineered antibody drug conjugate is purified and desalted by elutionthrough G25 resin in PBS, filtered through 0.2 μm filters under sterileconditions, and frozen for storage.

By the procedure above, cysteine engineered, antibody drug conjugates101-133 of Table 3 were prepared.

Example 21 In Vitro Cell Proliferation Assay

Efficacy of ADC was measured by a cell proliferation assay employing thefollowing protocol (CELLTITER GLO™ Luminescent Cell Viability Assay,Promega Corp. Technical Bulletin TB288; Mendoza et al (2002) Cancer Res.62:5485-5488):

-   1. An aliquot of 100 μl of cell culture containing about 10⁴ cells    (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each    well of a 96-well, opaque-walled plate.-   2. Control wells were prepared containing medium and without cells.-   3. ADC was added to the experimental wells and incubated for 3-5    days.-   4. The plates were equilibrated to room temperature for    approximately 30 minutes.-   5. A volume of CELLTITER GLO™ Reagent equal to the volume of cell    culture medium present in each well was added.-   6. The contents were mixed for 2 minutes on an orbital shaker to    induce cell lysis.-   7. The plate was incubated at room temperature for 10 minutes to    stabilize the luminescence signal.-   8. Luminescence was recorded and reported in graphs as RLU=relative    luminescence units.-   Data are plotted as the mean of luminescence for each set of    replicates, with standard deviation error bars. The protocol is a    modification of the CELLTITER GLO™ Luminescent Cell-   Media: SK-BR-3 grow in 50/50/10% FBS/glutamine/250 g/mL G-418    OVCAR-3 grow in RPMI/20% FBS/glutamine

Example 22 Tumor Growth Inhibition, In Vivo Efficacy in High ExpressingHER2 Transgenic Explant Mice and Other Tumor Models

Tumors were established and allowed to grow to 150-200 mm³ in volume (asmeasured using calipers) before a single treatment on day 0. Tumorvolume was measured using calipers according to the formula: V(mm³)=0.5A×B², where A and B are the long and short diameters,respectively. Mice were euthanized before tumor volume reached 3000 mm³or when tumors showed signs of impending ulceration. Data collected fromeach experimental group (10 mice per group) were expressed as mean±SE.

The Fo5 mouse mammary tumor model was employed to evaluate the in vivoefficacy of antibody-drug conjugates of the invention after single doseintravenous injections, and as described previously (Phillips G D L, LiG M, Dugger D L, et al. Targeting HER2-Positive Breast Cancer withTrastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate. (2008) CancerRes. 68:9280-90), incorporated by reference herein. Anti-Her2 ADC weretested with the Fo5 model, a transgenic mouse model in which the humanHER2 gene is over-expressed in mammary epithelium under transcriptionalregulation of the murine mammary tumor virus promoter (MMTV-HER2) asshown in FIGS. 31 and 32. The HER2 over-expression causes spontaneousdevelopment of a mammary tumor. The mammary tumor of one of thesefounder animals (founder #5 [Fo5]) has been propagated in subsequentgenerations of FVB mice by serial transplantation of tumor fragments(˜2×2 mm in size). All studies were conducted in accordance with theGuide for the Care and Use of Laboratory Animals. Each antibody-drugconjugate (single dose) was dosed in nine animals intravenously at thestart of the study, and 14 days post-transplant. Initial tumor size wasabout 200 mm³ volume. Measurements of tumor growth inhibition over timeby antibody-drug conjugates of the invention and controls are shown inFIGS. 31-34.

The OVCAR-3 mammary fat pad transplant efficacy model was employed asdescribed (Chen et al. (2007) Cancer Res 67:4924-4932), evaluating tumorvolume after a single intravenous dose and using tumors excised from amouse bearing an intraperitoneal tumor, then serially passaged into themammary fat pads of recipient mice (FIG. 33).

The efficacy of the anti-Napi2B antibody-drug conjugates (ADCs) wasinvestigated in a mouse xenograft model of Igrov-1 (human ovariancancer).

Female C.B-17 SCID-beige mice (Charles River Laboratories; San Diego,Calif.) were each inoculated in the thoracic mammary fat pad area with 5million Igrov-1 cells. When the xenograft tumors reached an averagetumor volume of 100-300 mm3 (referred to as Day 0), animals wererandomized into groups of 7-10 mice each and received a singleintravenous injection of the ADCs. Tumors and body weights of mice weremeasured 1-2 times a week throughout the study. Mice were promptlyeuthanized when body weight loss was >20% of their starting weight. Allanimals were euthanized before tumors reached 3000 mm3 or showed signsof impending ulceration. The anti-Napi2B antibody-drug conjugate (ADC134) demonstrated dose-dependent inhibition of tumor growth comparedwith vehicle group. The non-targeting control (ADC 137) had no effect ontumor growth (FIG. 36).

The efficacy of the anti-CD33 antibody-drug conjugates (ADCs) wasinvestigated in a mouse xenograft model of HL-60 or EOL-1 (human acutemyeloid leukemia). The HL-60 cell line was obtained from ATCC (AmericanType Culture Collection; Manassas, Va.) and EOL-1 cell line wasoriginated from DSMZ (German Collection of Microorganisms and CellCultures; Braunschweig, Germany).

Female C.B-17 SCID mice (Charles River Laboratories; Hollister, Calif.)were each inoculated subcutaneously in the flank area with five millioncells of HL-60 or EOL-1. When the xenograft tumors reached an averagetumor volume of 100-300 mm3 (referred to as Day 0), animals wererandomized into groups of 7-10 mice each and received a singleintravenous injection of the ADCs. Approximately 4 hours prior toadministration of ADCs, animals were dosed intraperitoneally with excessamount (30 mg/kg) of anti-gD control antibody to block possiblenonspecific antibody binding sites on the tumor cells. Tumors and bodyweights of mice were measured 1-2 times a week throughout the study.Mice were promptly euthanized when body weight loss was >20% of theirstarting weight. All animals were euthanized before tumors reached 3000mm3 or showed signs of impending ulceration.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. All patents, patent applications,and references cited throughout the specification are expresslyincorporated by reference.

We claim:
 1. An antibody-drug conjugate compound having the formula:Ab-(L-D)_(p) wherein: Ab is an anti-Napi2b antibody; L is a linkerhaving the formula:-Str-(Pep)_(m)-(Sp)_(n)- where Str is a stretcher unit covalentlyattached to the anti-Napi2b antibody; Pep is an optional peptide unit oftwo to twelve amino acid residues, Sp is an optional spacer unitcovalently attached to a dimer drug moiety, and m and n areindependently selected from 0 and 1; p is an integer from 1 to 8; D isthe dimer drug moiety having the formula:

where R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L;R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to L; R^(a)and R^(b) are independently selected from H and C₁-C₆ alkyl optionallysubstituted with one or more F, or R^(a) and R^(b) form a five or sixmembered heterocyclyl group; T is a tether group selected from C₃-C₁₂alkylene, Y, (C₁-C₆ alkylene)-Y-(C₁-C₆ alkylene), (C₁-C₆alkylene)-Y-(C₁-C₆ alkylene)-Y-(C₁-C₆ alkylene), (C₂-C₆alkenylene)-Y-(C₂-C₆ alkenylene), and (C₂-C₆ alkynylene)-Y-(C₂-C₆alkynylene); where Y is independently selected from O, S, NR¹, aryl, andheteroaryl; where alkylene, alkenylene, aryl, and heteroaryl areindependently and optionally substituted with F, OH, O(C₁-C₆ alkyl),NH₂, NHCH₃, N(CH₃)₂, OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl isoptionally substituted with one or more F; or alkylene, alkenylene,aryl, and heteroaryl are independently and optionally substituted with abond to L; D′ is the drug moiety having the formula:

where the wavy line indicates the site of attachment to T; X¹ isselected from O and NR³, where R³ is selected from H and C₁-C₆optionally substituted with one or more F; R⁴ is H, CO₂R, or a bond toL, where R is C₁-C₆ alkyl or benzyl; and R⁵ is H or C₁-C₆ alkyl; and theanti-Napi2b antibody comprises three light chain hypervariable regions(HVR-L1, HVR-L2 and HVR-L3) and three heavy chain hypervariable regions(HVR-H1, HVR-H2 and HVR-H3) wherein: HVR-L1 comprises the amino acidsequence of SEQ ID NO:75; HVR-L2 comprises the amino acid sequence ofSEQ ID NO:76; HVR-L3 comprises the amino acid sequence of SEQ ID NO:77;HVR-H1 comprises the amino acid sequence of SEQ ID NO:78; HVR-H2comprises the amino acid sequence of SEQ ID NO:79; and HVR-H3 comprisesthe amino acid sequence of SEQ ID NO:80 or the anti-Napi2b antibodycomprises a light chain hypervariable region comprising the amino acidsequence of SEQ ID NO:81 or a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:82.
 2. The antibody-drug conjugatecompound of claim 1 wherein Str has the formula:

wherein R⁶ is selected from the group consisting of C₁-C₁₀ alkylene,C₃-C₈ carbocyclyl, O-(C₁-C₈ alkyl), arylene, C₁-C₁₀ alkylene-arylene,arylene-C₁-C₁₀ alkylene, C₁-C₁₀ alkylene-(C₃-C₈ carbocyclyl), (C₃-C₈carbocyclyl)-C₁-C₁₀ alkylene, C₃-C₈ heterocyclyl, C₁-C₁₀ alkylene-(C₃-C₈heterocyclyl), (C₃-C₈ heterocyclyl)-C₁-C₁₀ alkylene, C₁-C₁₀alkylene-C(O)N(R⁸)—C₂-C₆ alkylene-N(R⁸), N(R⁸)—(C₂-C₆ alkylene), and(CH₂CH₂O)_(r)—CH₂: where R⁸ is H or C₁-C₆ alkyl, and r is an integerranging from 1 to
 10. 3. The antibody-drug conjugate compound of claim 2wherein R⁶ is (CH₂)₅.
 4. The antibody-drug conjugate compound of claim 1wherein m is 0 and n is
 0. 5. The antibody-drug conjugate compound ofclaim 1 wherein m is 0 and n is
 1. 6. The antibody-drug conjugatecompound of claim 1 wherein Str has the formula:

wherein R⁷ is selected from C₁-C₁₀ alkylene, C₁-C₁₀ alkylene-O,N(R⁸)—(C₂-C₆ alkylene)-N(R⁸), N(R⁸)—(C₂-C₆ alkylene), and(CH₂CH₂O)_(r)—CH₂; where R⁸ is H or C₁-C₆ alkyl, and r is an integerranging from 1 to
 10. 7. The antibody-drug conjugate compound of claim 1wherein Str has the formula:

wherein R⁷ is selected from C₁-C₁₀ alkylene, C₁-C₁₀ alkylene-O, (C₂-C₆alkylene)-N(R⁸), and (CH₂CH₂O)_(r)—CH₂; where R⁸ is H or C₁-C₆ alkyl,and r is an integer ranging from 1 to
 10. 8. The antibody-drug conjugatecompound of claim 7 wherein L forms a disulfide bond with a cysteineamino acid of the antibody, and R⁹ is C₂-C₆ alkylene-O where alkylene isoptionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃, N(CH₃)₂,OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally substituted withone or more F.
 9. The antibody-drug conjugate compound of claim 1wherein m is 1 and n is
 1. 10. The antibody-drug conjugate compound ofclaim 1 wherein m is 1 and Pep comprises two to twelve amino acidresidues independently selected from glycine, alanine, phenylalanine,lysine, arginine, valine, and citrulline.
 11. The antibody-drugconjugate compound of claim 10 wherein Pep is valine-citrulline.
 12. Theantibody-drug conjugate compound of claim 1 wherein n is 1 and Spcomprises para-aminobenzyl or para-aminobenzyloxycarbonyl.
 13. Theantibody-drug conjugate compound of claim 9 having the formula:

where AA1 and AA2 are independently selected from an amino acid sidechain; p is an integer from 1 to
 8. 14. The antibody-drug conjugatecompound of claim 13 wherein the amino acid side chain is independentlyselected from H, —CH₃, —CH₂(C₆H₅), —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂NHC(NH)NH₂, —CHCH(CH₃)CH₃, and —CH₂CH₂CH₂NHC(O)NH₂.
 15. Theantibody-drug conjugate compound of claim 13 having the formula:


16. The antibody-drug conjugate compound of claim 13 having the formula:


17. The antibody-drug conjugate compound of claim 15 having the formula:


18. The antibody-drug conjugate compound of claim 16 having the formula:


19. The antibody-drug conjugate compound of claim 18 having the formula:


20. The antibody-drug conjugate compound of claim 16 having the formula:


21. The antibody-drug conjugate compound of claim 20 having the formula:


22. The antibody-drug conjugate compound of claim 16 having the formula:

where R⁷ is independently selected from H and C₁-C₁₂ alkyl.
 23. Theantibody-drug conjugate compound of claim 1 where R^(a) and R^(b) form afive or six membered heterocyclyl group selected fromN-methylpiperazinyl, morpholinyl, piperidyl, and pyrrolidinyl.
 24. Theantibody-drug conjugate compound of claim 1 where T is C₃-C₅ alkylene.25. The antibody-drug conjugate compound of claim 24 where T is selectedfrom (CH₂)₃, (CH₂)₄ and (CH₂)₅.
 26. The antibody-drug conjugate compoundof claim 1 where T is (C₁-C₆ alkylene)-Y-(C₁-C₆ alkylene), where Y isphenyl substituted with a bond to L.
 27. The antibody-drug conjugatecompound of claim 1 where T is (C₂-C₆ alkenylene)-Y-(C₂-C₆ alkenylene),where Y is phenyl substituted with a bond to L.
 28. The antibody-drugconjugate compound of claim 1 where Y is selected from phenyl, pyridyl,1-methyl-1H-benzo[d]imidazole, and [1,2,4]triazolo[1,5-a]pyridine.
 29. Apharmaceutical composition comprising the antibody-drug conjugatecompound of claim 1, and a pharmaceutically acceptable carrier, glidant,diluent, or excipient.